INTRODUCTION:

Free radicals or ROS (Reactive Oxygen Species) are generated during normal metabolic process, they are extremely reactive, highly unstable and potentially damaging transient chemical species. Physiologically cellular redox state is controlled by scavengers like endogenous enzymes and exogenous enzymes from diet and also by some hormones. However imbalance between overproduction of free radicals and deficiency of enzymatic antioxidants produces oxidative stress on the physiological system. The excess oxidative stress can cause damage to cellular lipids, proteins or DNAwhich further inhibit their normal functions.

This oxidative stress plays crucial role in pathogenesis of many chronic degenerative diseases such as atherosclerosis, coronary heart diseases, aging and cancer¹. Oxidative stress also contributing to conditions like adult respiratory distress syndrome, rheumatoid artheritis, Diabetes mellitus, liver disorder, Parkinsons disease, Alzhemers and AIDSand also responsible for aging^2,3.

So if attention is given towards minimizing free radical generated oxidative stress which will automatically improve physical conditions as well as prevent degenerative diseases which are results due to formation of free radicals. Antioxidant are the substance that prevent the tissue damage results due to free radicals. Antioxidants are widely used in pharmaceutical industry and food industry as they prolongs self life of food products. At present there are many more synthetic antioxidants are used in food industry but some of them having toxic effects mainly carcinogenic¹. Therefore there are necessities to search new natural antioxidants which is safe and cost effective as it will be widely used in pharmaceutical preparations for therapeutic application and food industry, and hence many researcher had develop interest to find natural antioxidant from plant products⁴.

Literature studies reveled that many researcher throught the world evaluated medicinal properties of plants for their therapeutic applications due to their antioxidant activities, no or minimal side effects and most important is that due to their cost effectiveness⁵. Interest in plant materials rich in polyphenolic compounds increases recently due to their high antioxidant potency^{6 .}Medicinal plants plays key role in prevention of many human health problems since far ago. According to world health organization (WHO) nearly 80% of population in Asian and African countries depends upon traditional medicines for primary health issues⁷. Bioactive compounds present in medicinal plant which could either delay or inhibit the starting of degenerative diseases and increases life expectancy of suffering individuals⁸.

Psidium guajava L.is a small tree or shrub from family myrtaceae commonly known as guava in English. Guava is native to South America but now found in several regions of tropical and subtropical countries of the world. It also known as ‘poor mans apple’ as it is widely and easy to grown even in backyard of houses. Psidium guajava is an important food crop and widely used traditional medicinal plant due to its diverse medicinal values for various ailmentsall around the world. Though fruit having nutritional values all parts of guava tree like fruits, leaves, barks and roots have been used in treatment of stomache and diarrhoea, whereas leaves and seeds are used in treatment of respiratory and GIT disorders and as an antispasmodic, anti-inflammatory, in cough remedies, in hypertension management, obesity and in the control of diabetes mellitus. Seeds of psidium guajava shows antimicrobial,gastrointestinal,antiallergic and anticarcinogenic activity^9,10,11. Guava also posseses wound healing property¹². Apart from its medicinal values guava fruit is also attraction of food industry to produce candies,juicefatty acids,jams and frozen pulp¹³.

Guava is rich in tannins, phenols, triterpines, flavonoids, essential oils, saponins, carotenoids, lectins, vitamins, fiber and fatty acids. Guava fruit is higher in vitamin C than citrus and also contain appreciate amount of vitamin A. Leaves of guava are rich in flavonoids particularly quercetin¹⁴. Among the all bioactive compounds of medicinal plants phenolic compounds has great value in developing new molecules as it posseses strong antioxidant activity and other activities like anti-inflammatory,antibacterial,antithrombotic¹⁵ and can be used as adjuant in treatment of obesity^16,17. Quercetin also has several pharmacological actions like anti-inflammatory, antiviral, antitumour and dose dependent antioxidant activity¹⁸. Phenolic compounds like flavonoids, tannins and phenolic acids are becoming wide interests for development of antioxidant product as phenolic compounds are free radical terminators.¹⁹ As psidium guajava leaves are rich in most of bioactive compounds mainly phenolic and flavonoid content and its availability is throught the world this plant becomes researchers interest to develop new molecule entity.

The objective of the present work was to quantify total phenolic compounds,total flavonoids and assessment of in vitro antioxidant capacities of aqueous and methanolic extracts obtained from leaves of Psidium guajava linn.

MATERIALS AND METHODS:

Plant collection and Authentication:

Fresh leaves of Psidium guajava L.were collected from the local area of Karad, Maharashtra,in the month of July 2018, and authenticated from botanical survey of India, Pune. voucher specimen (BSI/WRC/IDN.CER./ 2018/H3/97) deposited in herbarium for further reference. Leaves were washed with clean water and dried under shade and grinded to form coarse powder. The powder is packed in paper bags and stored in air tight container until use.

Preparation of Extracts:

Aqueous extract (AEPG):²⁰

100 gms of coarse powder is mixed with water and chloroform (9:1) and kept in glass container for 7 days with intermittent occasional shaking of container. After 7 days mixture is filtered through clean sterile musclin cloth and filtrate is again passed through whatsman filter paper No 1 .Filterate is boil to evaporate water. Brown colour residue is obtained after evaporation which is stored in air tight container and labeled as AEPG (Aqueous extract of psidium guajava).

Methanolic extract (MEPG):

100 gms of coarse powder of psidium guajava L was defated by petroleum ether (60-80°C) residue was allowed to dry at room temperature and then extracted with 200ml of 95% methanol in a soxhlet extractor for 72 hours.Then the extract collected and subjected to solvent evaporation. The dried green coloured extract was obtained which is stored in air tight container and labeled as MEPG (Methanolic extract of psidium guajava).

Chemicals:

1,1-diphenyl-2-picrylhydrazide (DPPH), (Sigma Aldrich, Ltd, India), Methanol, Aluminium chloride, Foline ciocalteu reagent, Sodiumcarbonate, Ascorbic acid, Sodium nitrite, Sodium hydroxide Gallic acid, Quercetin (All from Loba Chemie Pvt, Ltd, India).

Instruments:

Shimdzu UV Visible spectrophotometer.

Preliminary Phytochemical Screening of Extracts:

Aqueous and methanolic extracts of Psidium guajava L. were subjected to qualitative phytochemical screening to find out the presence of various phytochemical constituents using standard methods^21,22.

Estimation of Total Phenolic compounds:

The estimation of total phenolic contents in plant extract was determined by Folin-Ciocalteu method using spectrophotometer. Ethanolic solution of the extract (test solution) in the concentration of 1mg/ml was used in the analysis. The reaction mixture consist of 1ml extract and 9ml of distilled water in a 25ml volumetric flask. 1ml of Folin Ciocalteu phenol reagent was added to the mixture and shaken vigorously. After 5 minutes, 10ml of 7% Sodium carbonate solution was added to the mixture. The volume was adjustedup to 25ml. Similarly a set of standard solutions of Gallic acid (200, 400, 600, 800 and 1000µg/ml) were prepared in similar manner. Absorbance of test and standard solutions were measured against the reagent blank at 550nm on spectrophotometer after the incubation period of 90 min at room temperature. The total phenolic content was expressed as mg of Gallic acid equivalent (GAE) per g of extract. The absorbance of test sample was performed in triplicate²³.

Estimation of Total Flavonoid contents^23,24:

Total flavonoid content was measured by the aluminium chloride colorimetric assay.

Briefly, the reaction mixture contains 1ml of extract and 4ml of distilled water in a 10ml volumetric flask. Add, 0.3ml of 5% sodium nitrite and after 5 minutes, 0.3ml of 10% aluminium chloride solution added. After 6 minutes, 2ml of 1M sodium hydroxide was added and diluted to 10ml with distilled water.

A similar set of reference standard solutions of quercetin (200, 400, 600, 800 and 1000μg/ml) were prepared. The absorbance for test and standard solutions were determined against the reagent blank at 510nm with spectrophotometer. The flavonoids in extracts were estimated using standard curve plotted by different concentration of quercetin (100-1000µg/ml ). The total flavonoid content was expressed as mg of quercetin equivalents (QE) per g of extract. The absorbance of test sample was performed in triplicate. Plant constituents like flavone and flavonoids are secondary metabolite products which posseses antioxidant and antiradical activity.²⁵

In –Vitro Antioxidant activity by DPPH free radical scavenging assay^26,27,28:

1,1-diphenyl-2-picrylhydrazyl (DPPH) molecule is characterized as a stable free radical by due to its delocalizing ability to spare electron over the molecule as a whole, so it does not dimerize, Due to delocalization of electron it gives rise to the deep purple color, When DPPH reacts with reducing agent then it looses this purple colour stoichyometrically with the number of electrons consumed which is measured by spectrophotometer at 517nm.The activity was evaluated by observing decrease in absorbance due to change in color.Higher the concentration of sample then free radical scavenging effect is also stronger.²⁹

The ability of compounds to scavenge DPHH radical was assessed using Ramanathan Sambath Kumar and Manzocco et.al.,1998 method with slight modification in it.Briefly 1ml extract of conc.(1000µg/ml) was mixed with3 ml DPPH (0.5mmol/L in methanol),the resultant absorbance was recorded at 517nm after 30 min incubation at 37°C. The percentage of scavenging activity was derived using the following formula.

Percentage of inhibition (%) =[(A control – A sample)/ A control] x 100

Where A control - absorbance of DPPH

A sample - absorbance reaction mixture (DPPH with Sample)

Statistical Analysis:

All experiments were carried out in triplicates. Results were reported as mean±standard deviation (SD).

RESULTS AND DISCUSSION:

Percentage Yield:

Percentage yield to be found in AEPG was 6.2% and MEPG was 11.66 % with respect to the total quantity of Psidium guajava leaves powder used for extraction.

Preliminary Phytochemical Screening:

Aqueous and methanolic extracts of Psidium guajava L. leaves shows presence of flavonoids, tannins, phenoliccompounds, saponins, where asmethanolic extract additionally shows presence of steroids and triterpenoids.

Estimation of Total Phenolic Contents (TPC) and total Flavonoid contents (TFC):

The total phenolic contents of AEPG and MEPG were measured by using Folin Ciocalteu reagent using gallic acid as standard. The Folin-Ciocalteu reagent is sensitive to reducing compounds including polyphenols, thereby producing blue colour upon reaction with phenolic contents of extract which is measured spectrophotometrically at 550nm at this wavelength max.absorption is observed and proportional to quantity of phenolic content in extract.TPC was expressed in terms of gallic acid equivalent. equation for standard curve is y = 0.0005x R²= 0.989 (Figure 1). The values for total phenolic contents were calculated by using standard curve for gallic acid and it was found to be 37.72mg GAE/g and 84.91mg GAE/g dry weight of extract for AEPG and MEPG respectively (Table 1). The amount of total phenolic content present in herbal plant depends on factors such as temperature, UV-light, nutrition available to the plant, and also genetic factors ³⁰.

Similarly, The total flavonoid contents of AEPG and MEPG was assessed by using aluminium chloride (ALCl₃) method using quercetin as standard reagent. TFC was expressed in terms of quercetin equivalent by plotting calibration curve for standard quercetin,with correlation coefficient (R²=0.989). (Figure 2).Total flavonoid contents in AEPG was found to be 67.37mg QE/g and for MEPG was found to be 86.75mg QE/g of dry weight of material (Table 1)

MEPG contains high concentration of phenolic compounds. TPC and antioxidant activity of plant extract have direct relation that plant extract which having high content of TPC posseses high antioxidant activity¹⁶.

Table 1: Results of Total Phenolic contents (TPC) and Total Flavonoid contents (TFC) of Psidium guajava leaves extracts

Sr. No.	Sample at conc (1000µg/ml	Total Phenolic Contents (mg GAE/g dry material)	Total Flavonoid contents (mg QE/g dry material)
01	AEPG	37.72 ± 0.14	67.37 ± 0.19
02	MEPG	84.91 ± 0.10	86.75 ± 0.17

Values are ± SD.(n =3)

Whereas, AEPG is Aqueous extract of Psidium guajava leaves

MEPG is Methanolic extract of Psidium guajavaleaves

GAE is Gallic acid equivalent

QE is Quercetin equivalent

R² values represented mean data set of n=3

Figure 1: Total phenolic content for standard gallic acid

R² values represented mean data set of n=3

Figure: 2 Total Flavonoid contents for standard Quercetin

In-vitro Antioxidant activity by using DPPH free radical scavenging method:

Antioxidant activity of Psidium guajava leaves extracts was estimated by using DPPH free radical scavenging method which is widely used and highly sensitive method among all methods used to asses in-vitro antioxidant activity²⁸. Ability of guava leaves extracts to donate hydrogen atom to DPPH radical is measured in form of absorbance of DPPH solution then this absorbance was used to calculate the percent inhibition. Ascorbic acid is used as standard.

Our study shows that DPPH radical scavenging capacity of AEPG and MEPG was 40 % and 56.66 % inhibition respectively. Results are presented in table no 2.Whereas,MEPG having strong antioxidant activity as compare to AEPG and it was near equal to that of standard ascorbic acid.

DPPH scavenging method is widely used method for assessing the antioxidant activity. Our study (Table 3) shows that DPPH radical scavenging capacity of both AEPG and MEPG are 40 % and 56.66% inhibition respectively.

Table 2: Percentage inhibition of Psidium guajava L.leaves extracts

Sample code (1000 µg/ml)	DPPHradical scavenging activity
Sample code (1000 µg/ml)	Absorbance at 517nm	% inhibition
Control (DPPH)	0.30
Standard Ascorbic acid	0.12	60
AEPG	0.18	40.00
MEPG	0.13	56.66

Where, AEPG is aqueous extract of Psidium guajava,

MEPG is methanolic extract of Psidium guajava.

CONCLUSION:

From the results of present investigation indicated that it could be concluded that methanolic extract of Psidium guajava L.leaves(MEPG) having higher concentration of total phenolic and total flavonoid contents as compare to aqueous extract and having stong capacity to scavenge free radicals. Guava plant posseses potent antioxidant activity.In addition phenolic compounds and flavonoids appear to be responsible for the antioxidant activity³¹.On the basis of the results obtained in this study guava leaf extract can be used in therapeutic and pharmaceutical application as a preventive measurement of diseases which are results due to free radical generation.

However further studies of this guava plant species should be directed to carry out isolation of bioactive compounds which attributed its antioxidant activity and carry out in vivo studies to evaluate its medicinal values to prepare a natural pharmaceutical product that will be beneficial for human health.

ACKNOWLEDGEMENT:

Authors are grateful to Hon’ble Chairman Dr. Suresh J. Bhosale and research fund allotment committee for providing financial assistance and facilities to carry out PhD research project.

CONFLICT OF INTEREST:

The authors have declare that they have no any conflict of interest regarding publication of this article.

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Received on 05.08.2021 Modified on 18.04.2022

Research J. Pharm. and Tech 2023; 16(3):1028-1032.

DOI: 10.52711/0974-360X.2023.00172