Comparative Evaluation of multiple extracts of Pericarp of

Luffa acutangula (L.) for its significant Antioxidant, Antibacterial and Antifungal activity

 

Shubhangi Kadu1,2*, Daksha Attarde1, Deepak Kulkarni3, Santosh Shelke3, Vilas Arsul

1M.G.V.’s Pharmacy College, Panchwati, Nashik, 422003, Maharashtra, India.

2Dr. Y. S. Khedkar College of Pharmacy, CIDCO, Aurangabad, 431003, Maharashtra, India.

3Srinath College of Pharmacy, Bajajnagar, Waluj, Aurangabad, 431136, Maharashtra, India.

*Corresponding Author E-mail: sskadu79@rediffmail.com

 

ABSTRACT:

Luffa acutangula (L.) is the commonly used vegetable in different parts of India which belongs to the family Cucurbetaceae. Pericarp of Luffa acutangula (L.) is generally considered as a waste part. The aim of the present investigation was to evaluate biological activities of the pericarp extracts of Luffa acutangula (L.) Flavonoids and phenolic compounds are widely distributed in plant which exerts multiple biological effects, including antioxidant, antibacterial and antifungal activity. Petroleum ether, chloroform and methanolic extracts of pericarp Luffa acutangula (L.) were used for comparative study of its antioxidant and antimicrobial property. Methanolic extract of pericarp shows highest amount of phenolic (45.0±1.1mg GAE/g of extract) and flavonoid content (4.5±0.2mg rutin/g of extract). Different models were used to study antioxidant properties of extract like 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity model, Nitric oxide scavenging activity model and H2O2 scavenging activity model and in most of the models, it was found that methanolic extract is having potent antioxidant property as compare to extracts of other solvents. Antibacterial activity was studied against strains of Escheria coli, Pseudomonas aeruginosa, Staphylococcus aureus and antifungal activity was studied against culture of Candida albicans. All three extracts of pericarp showed significant antimicrobial activity. Evaluation of this therapeutic potential of phytochemicals present in pericarp shows its great medicinal importance.

 

KEYWORDS: Luffa acutangula (L.), Antioxidant, Phytochemical, Antibacterial, Antifungal, Scavenging activity.

 

 


INTRODUCTION:

Reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide and hydroxyl, nitric oxide radicals, play an important role in oxidative stress related to the pathogenesis of various important diseases1. Antioxidants act as a major defense against free oxygen radical mediated toxicity2. In past few years researchers have a great deal of research interest in natural antioxidants.  Antioxidant research is an important topic in the medical field as well as in the food industry.

 

 

These Recent research about important bioactive compounds in many plants, vegetables and food materials has received much attention3. The oxidation induced by ROS (Reactive Oxygenated Species) can result in cell membrane disintegration, membrane protein damage and DNA mutation, which can further initiate or propagate the development of many diseases, such as cancer, liver injury and cardiovascular disease, although the body possesses such defense mechanisms, as enzymes and antioxidant nutrients, which arrest the damaging properties of ROS. Therefore, bioactive plants with free radical scavenging activities have great relevance in the prevention and therapeutics of diseases in which oxidants or free radicals are causative factors4. In this respect, polyphenolic compounds, like flavonoids and phenolic acids, commonly found in plants have been reported to have multiple biological effects, including antioxidant activity5.

Cucurbitaceae family is well known to all of us as a good source of vegetable, which has health benefits. The family cucurbitaceae consists of about 117 genera and 825 species out of which about 15 different species of cucurbitaceae are being cultivated in Asia6. They have numerous resemblances in gourd development. Some well-known vegetables fall in this family such as Watermelon, Butternut, Pumpkin, sweet melons and Cucumber, gourds, melons, pumpkins and squashes. They are characterized by their fleshy fruits7. The seeds of many species of the group have been noted for their oil bearing characteristics. The Cucurbita genus (squash) is said to have evolved in the warmer parts of the Americas. This family in relation with nutraceuticals provides natural antioxidant as it contains polyphenolics, flavonoids, flavanols, and carotenoids and may give antioxidant activity8.

 

Antibacterial and antifungal activity of drug can be determined in vitro with the help of different cultures of bacteria and fungi. Minimum Inhibitory Concentration (MIC) is the quantitative terminology which describes the minimum amount of substance required to inhibit the growth of microorganism. Multiple plant species from cucurbetacea exhibits antimicrobial properties9. Zone of growth inhibition is usually measured to determine antimicrobial property of crude extract which is compared with different antibiotics as a standard10.

 

MATERIAL AND METHODS:

Plant material and chemicals:

Sample of Luffa acutangula (L.) Was collected in the month of December from local market. Herbarium was prepared and authentication was done from Botanical Survey of India-Pune, after the authentication of plant it was used for further studies. All remaining chemicals were brought from Qualigens fine chemicals, Mumbai.

 

Morphological and microscopic evaluation:

Pericarp of Luffa acutangula (L.) Was studied for its morphological characteristics. Along with morphology, microscopic evaluation is also carried out for that The Pericarp was cut in thin section and fixed in FFA (Formalin-5ml + Acetic acid-5ml +70% +Ethyl alcohol-90ml). After 24 hrs of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol. Infiltration of the specimen was carried by gradual addition of paraffin wax tertiary butyl alcohol solution attains supersaturation. The specimens were cast into paraffin blocks. The paraffin embedded specimen was sectioned with the help of rotary microtome. The thickness of the section was 10-12 mm. Dewaxing of the section was done and that was stained using Phloroglucinol HCl and mounted under microscope and observed for microscopic characteristics11.

 

Determination of ash values and moisture content:

2g accurately weighed powdered pericarp of; Luffa acutangula (L.) Was incinerated in silica crucibles at a temperature of 45000C in muffle furnace, until it was free from carbon, cooled and weighed. The percentage of ash was calculated with reference to the air-dried powder. Water soluble, insoluble and acid soluble, insoluble as well as sulphated ash value was determined12.

 

For moisture content determination about 10g of pericarp of Luffa acutangula (L.), without preliminary drying and also dried powder of pericarp was placed in a tared evaporating dish. After placing the above said amount of the plant in the tared evaporating dish, it was dried at 1050 for 5 hours, and weighed. Drying and weighing was continued at one hour interval until difference between two successive weighing correspond to not more than 0.25 %13.

 

Phytochemical extraction:

Pericarp of Luffa acutangula (L.) Was removed, dried in shade, powdered and used for extraction. 245.20g powder of Luffa acutangula (L.) Was extracted using methanol, Petroleum ether and Chloroform as a solvent. Extract obtained was kept in desiccator and yield extractive values were calculated with respect to its weight. Phytochemical tests were carried out to find organic as well as inorganic chemical constituents present in the petroleum ether, chloroform and methanolic extracts of pericarp of Luffa acutangula (L.)14.

 

Phytochemical screening:

Extracts of Luffa acutangula (L.) Was subjected for phytochemical screening for different constituents like carbohydrates, amino acids, proteins, steroids, glycosides, alkaloids, tannins, polyphenolic compounds, flavonoids and phytosterols15.

 

Total phenolic content in extract:

From standard stock solution (1000μg/ml) of gallic acid different concentrations were prepared as 10, 20, 30, 40, 50, 75, 100μg/ml with water, 1ml of Folin Ciocalteu reagent (FCR) (1:2) was added in each concentration and incubated for 10 min. After that 4ml sodium carbonate was added and finally incubated for 2 hr (keep in dark place) and absorbances were recorded against blank at 740nm on ultravoilet (UV) spectrophotometer (Shimadzu 1800). Standard blank- prepared without using extract. For methanolic extracts concentration were taken as 50μg/ml from the stock solution of Luffa acutangula (L.) And remaining procedure was followed as same as standard16.

 

Total flavonoid content in extract:

Total flavonoid content was measured by the aluminum chloride assay. 1ml of extracts or standard solution of rutin (500μg/ml) was added to 10ml volumetric flask containing 4ml of water. To the flask was added 0.3ml 5 % sodium nitrite. After 5 min, 0.3ml 10 % aluminium chloride was added. At 6th min, 2ml 1M sodium hydroxide was added and the total volume was made up to 10ml with water. The solution was mixed well and the absorbance was measured against prepared reagent blank at 510nm. Total flavonoid content of extracts was expressed as total mg rutin/g of extract. Samples were analysed in triplicates17.

 

Evaluation of invitro antioxidant properties of extract:

In vitro antioxidant property of pricarp of Luffa acutangula (L.) Was studied for all three extracts (petroleum ether, chloroform and methanolic) using different scavenging activity models.

 

DPPH (2, 2-Diphenyl-1-picrylhydrazyl) scavenging activity:

DPPH scavenging activity is one of the common methods of antioxidant property determination. Total DPPH scavenging activity of pericarp of Luffa acutangula (L.) Was determined using ascorbic acid as a standard and DPPH (2, 2-Diphenyl-1-picrylhydrazyl) as a reagent. Standard ascorbic acid solution (1500μg/ml) was prepared. Sample solution was prepared by dissolving 15mg of petroleum ether, chloroform and methanolic extract in 10ml methanol (1500μg/ml). 1 ml standard of different concentrations was made in methanol from the stock solution (10, 20, 30, 40, 50μg/ml), then 1ml methanol and 1 ml DPPH was added in each concentration, absorbance was recorded after 15 min at 517nm. Control was prepared without using extract. Same procedure was applied for each extracts and concentrations used were 10, 50, 100, 150, 200, 300, 500μg/ml and absorbance was recorded. Scavenging of the DPPH free radical was measured by using the following formula and graph of comparative study was plotted18,19.

 

% Inhibition =Ablank – Astandard/ Ablank × 100

 

Nitric oxide scavenging activity:

Nitric oxide scavenging activity is one of the methods of antioxidant property determination. Total Nitric oxide scavenging activity of pericarp of Luffa acutangula (L.) Was determined using ascorbic acid as standard and Griess reagent. Standard ascorbic acid solution (1000 μg/ml) was prepared and different dilutions were made in range of 50-300μg/ml, absorbance was recorded. Sample solution was prepared by dissolving 10mg of petroleum ether, chloroform and methanolic extract of both the plants in 10ml alcohol (1000μg/ml). 1ml of different concentrations (50, 100, 150, 200, 250, 300 μg/ml) of petroleum ether, chloroform and methanolic extracts of plant as well as ascorbic acid was added to different test tubes and incubated for 150 min with addition of 2ml (10mm) sodium nitroprusside, from above stock solution of each concentration, 0.5ml of reaction mixture was taken, to it 2ml Griess reagent was added with interval of 5 min in between them and incubated for 30 min. A pink colored chromophore is formed. The absorbance of these solutions was measured at 540nm20, 21.

 

Hydrogen peroxide (H2O2) scavenging activity:

Hydrogen peroxide (H2O2) scavenging activity method was also used for antioxidant property determination of Luffa acutangula (L.). There are two methods for this determination first is ultraviolet spectroscopic method and second is titrimetric method. In ultraviolet method ascorbic acid was used as standard and H2O2 as reagent. Standard ascorbic acid solution (5000μg/ml) was prepared. Sample solution was prepared by dissolving 50mg of petroleum ether, chloroform and methanolic extract of both the plants in 10ml water (5000μg/ml). Solution of H2O2 (40mm/L-1) is prepared in phosphate buffer (7.4), concentration range is taken from 250 -2500μg/ ml then 0.6ml of H2O2 reagent was added. After 10 min, absorbance was recorded at 230nm and same procedure was repeated with samples and absorbance was recorded22.

 

For titrimetric determination of H2O2 scavenging activity, 0.1mm H2O2was prepared, from that 1ml was taken, concentrations were prepared in range from 1000- 1500μg/ml of both standard as well as all three extracts of plant. Further two drops of 3% of ammonium molybdate and 10ml of 2M sulphuric acid and 7ml of 1.8M of potassium iodide were added and titrated with 5.09mm of sodium thiosulphate, until yellow color disappears. Absorbance for different concentrations and standard ascorbic acid was recorded23.

 

Antibacterial activity of extract

Antibacterial activity of all three extracts of pericarp of Luffa acutangula (L.) Was studied against the strains of Escherichia coli (E. coli) (Gram negative bacteria), Pseudomonas aeruginosa (Gram positive bacteria), and Staphylococcus aureus (Gram positive bacteria). Gentamicin was used as standard for to compare with all three extracts24.

 

Procedure for antibacterial activity testing:

Plates were prepared with Muller-Hinton agar and used for Kirbey-Bauer dilution method. Medium plate was sterilized and depth 4 mm was kept. From the pure culture 3-4 similar colonies were transferred to about 5ml of tryptone soya broth and incubated 350C for 2-8 hours. Wooden applicator with non toxic cotton swab was dipped in inoculum streaked over a surface plate for three times. Deposit octodiscs at the centre of plate. The plate was incubated at 370C and examined after 19 hours. Zones showing complete inhibition were observed and diameters of zones were recorded25.

 

Antifungal activity of extract:

Antifungal activity of pericarp extract was studied against the strains of Candida albicans, using Ketoconazole as a control standard.

 

Procedure for antifungal activity testing:

Plates were prepared with Sabouraud dextrose agar for growing Candida albicans. Same procedure was carried out which was done for testing of antibacterial activity. The plate was incubated at 25-300C and examined after 48 hours. Zones showing growth inhibition were observed and diameters of zones were recorded26.

 

RESULTS AND DISCUSSION:

Microscopic evaluation of pericarp:

The transverse section of Luffa acutangula (L.) Pericarp is having three zones i.e., Epicarp with single layer, thickened, polygonal cells, mesocarp with parenchymatous cells and vascular bundles and endocarp with thick walled polygonal cells27.

 

Ash values and moisture content of pericarp:

Total ash value of pericarp of Luffa acutangula (L.) Was found to be 6.5% w/w, water soluble ash 4% w/w, acid insoluble 3% w/w and sulphated ash 3% w/w. LOD (Loss on Drying) of fresh pieces and pericarp was found to be 80.5% w/w and 10.7% w/w respectively28.

 

Extractive values and phytochemical screening:

Phytochemical analysis was done for petroleum ether, chloroform and methanolic extracts of Luffa acutangula (L.) The extractive values for petroleum ether, chloroform and methanolic extracts were 2.02%, 1.33% and 23.40% w/w respectively. Consistency of petroleum ether and chloroform extract was solid while methanolic extract is semisolid. In petroleum ether extract only steroids gives positive test with Liebermann Burchard reagent. In chloroform extracts glycosides are present as active chemical constituents. In methanolic extract carbohydrates, reducing sugars, flavonoids, tannins and phenolic compounds, amino acids and proteins present as a chemical constituents (Table 1)29.

 

Table 1: Phytochemical screening of all three extracts of pericarp of Luffa acutangula (L.)

Sr. No.

Phytochemical constituents

Petroleum ether extract

Chloroform extract

Methanol extract

1

Carbohydrate

­­-

-

+

2

Reducing sugars

­­-

-

+

3

Monosaccharides

­­-

-

+

4

Non reducing polysaccharides

-

-

-

5

Gums

-

-

-

6

Mucilage

-

-

-

7

Amino acids

­­-

-

+

8

Proteins

­­-

-

+

9

Steroids

+

-

-

10

Glycosides

-

+

+

11

Flavonoids

­­-

-

+

12

Alkaloids

-

-

-

13

Tannins and phenolic compounds

­­-

-

+

+ = Present and – = Absent

 

Total Polyphenolic content of Methanolic extract as equivalent of gallic acid by using Folin ciocaltea reagent (FCR) was found to be 45.0±1.1mg GAE/g of extract for Luffa acutangula (L.). The total flavonoid content in methanolic extract of Luffa acutangula (L.) Was found to be 4.5±0.2mg rutin/g of extract30.

 

Antioxidant activities of extracts:

Pericarp extracts of Luffa acutangula (L.) Contains significant proportion of phytochemicals like total phenolic contents and flavonoid contents. Presence of these chemical constituents significantly contributes for antioxidant activity of extract.

 

DPPH scavenging activity:

In DPPH scavenging activity method, the IC50 of methanolic extract of pericarp Luffa acutangula (L.) Was found to be 213.59±1.50μg/ml which having potent antioxidant as compared to petroleum ether and chloroform extract which are having IC50 as 366.77±2.20 and 260.78 ±1.79μg/ml respectively (Fig. 1) (Table 2)31.

 

Fig. 1: Comparative DPPH scavenging activity of all three extracts of pericarp of Luffa acutangula (L.)

 

Table 2: Comparative Inhibitory Concentration (IC50) of all three extracts of pericarp of Luffa acutangula (L.)

                                                            IC50 (µg/ml)

Sr. No.

In vitro antioxidant model

Petroleum ether extract

Chloroform Extract

Methanolic Extract

1

DPPH scavenging activity

366.77±

2.20

260.78 ±

1.79

213.59 ±1.50

2

Nitric oxide scavenging activity

223.22±

2.50

237.70 ±

2.15

189.01±

1.10

3

H2O2 scavenging activity by UV method

458.60±

2.90

373.26±

1.40

252.00 ±1.65

 

4

H2O2 scavenging activity by titrimetric method

808.40±

3.50

625.00±

2.78

533.71±

3.22

Results are expressed as means ± standard deviation (n=3)

 

Nitric oxide scavenging activity:

Methanolic extract of pericarp also showed better scavenging activity against nitric oxide with IC50 of 189.01±1.10, as compare to remaining two extracts (Fig. 2) (Table 2).

 

 

Fig. 2: Comparative Nitric oxide scavenging activity of all three extracts of pericarp of Luffa acutangula (L.)

 

Hydrogen peroxide (H2O2) scavenging activity:

In Hydrogen peroxide (H2O2) scavenging activity by UV method for Luffa acutangula (L.), the IC50 of Petroleum ether and Chloroform extract was 458.60±2.90 and 373.26±1.40μg/ml and for Methanolic extract was found to be 252.00 ±1.65μg/ml and by titrimetric method the IC50 of Petroleum ether and Chloroform extract was 808.40±3.50 and 625.00±2.78μg/ml and Methanolic extract was found to be 533.71±3.22μg/ml (Fig. 3 and 4) (Table 2)32.

 

 

Fig. 3: Comparative H2O2 scavenging activity of all three extracts of pericarp of Luffa acutangula (L.) By UV method.

 

 

Fig. 4: Comparative H2O2 scavenging activity of all three extracts of pericarp of Luffa acutangula (L.) By titrimetric method.

 

Correlation between total phenolic content and scavenging activity of methanolic extract was found to be relevant. Methanolic extract of pericarp was found to have more amounts of phenolic and flavonoid content as compare to remaining two extracts. Phenolic constituents and flavonoids produces significant scavenging activity against reactive oxygen species and reduce oxidative stress so methanolic extract was to be more efficient in antioxidant activity as compare to petroleum ether and chloroform extracts32.

 

Antibacterial and Antifungal activity of extracts:

Antibacterial and antifungal activities of all three extracts of pericarp of Luffa acutangula (L.) Were seen against different aerobic species bacteria and species of fungi respectively. Zone of growth inhibition of methanolic extract was found to be significant in comparison with Gentamicin which was used as a standard (Table 3).

 

Table 3: Antibacterial activity study of all three extracts of pericarp of Luffa acutangula (L.)

 

Zone of growth inhibition (mm)

Sr. No.

Sample

E. coli

Pseudomonas aeruginosa

Staphylococcus aureus

1

Petroleum ether extract

09.0±0.5

07.5±0.3

08.1±0.4

2

Chloroform extract

08.7±0.4

09.2±0.2

07.7±0.3

3

Methanolic extract

13.0±0.8

12.6±0.4

12.9±0.5

4

Gentamicin

20.0±0.5

21.2±1.0

22.2±0.7

Results are expressed as means ± standard deviation (n=3)

 

Similarly, in antifungal activity methanolic extract showed significant growth inhibition against the strain in comparison with Ketoconazole (Table 4).  Phenolic and flavonoid contents are responsible for antimicrobial, antifungal activities compounds so methanolic extract of pericarp is found to be better as compare to other two extracts (Figure 5)33.

 

Table 4: Antifungal activity for all three extracts of pericarp of Luffa acutangula (L.) Against strains of candida albicans.

Sr. No.

Sample

Zone of growth inhibition (mm)

1

Petroleum ether extract

09.1±0.5

2

Chloroform extract

08.9±0.4

3

Methanolic extract

13.6±0.6

4

Ketoconazole USP

23.2±0.9

Results are expressed as means ± standard deviation (n=3)

 

Fig. 5: Comparative antibacterial and antifungal activity of all three extracts of pericarp of Luffa acutangula (L.) method.

 

CONCLUSION:

This research investigated the significant antioxidant and antimicrobial property of pericarp of Luffa acutangula (L.). Antioxidant activity was studied with DPPH, nitric oxide and H2O2 scavenging activity models and it is concluded that methanolic extract of pericarp have better antioxidant property. Antimicrobial activity was studied with different strains of gram positive and gram negative bacteria using Gentamicin as a standard. All three extract showed significant antibacterial activity. Pericarp extracts also shown better antifungal activity against candida albicans in comparison Ketoconazole as standard. This significant antioxidant and antimicrobial property of pericarp of Luffa acutangula (L.) creates opportunity for commercial dosage form development and inclusion pericarp as a part of diet.

 

ACKNOWLEDGEMENT:

The authors sincerely acknowledge the support of MGV’s College of Pharmacy, Panchwati Nashik for providing all the amenities and environment to execute this work. Authors also sincerely thank the Botanical Survey of India, Pune for and authentication of plant.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 06.05.2020            Modified on 11.07.2020

Accepted on 22.08.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):1847-1853.

DOI: 10.52711/0974-360X.2021.00327