Phytochemical screening for Antioxidant and Antifungal activity of Lagenaria siceraria (Molina) Standl. leaf extract

 

Tarange R G1*, Kharat J S2, Jedage H D3, Manjunath K P4

1,3Department of Pharmacognosy, L. N. B. Chhabada Institute of Pharmacy (Degree), Raigaon, Satara - 415020, (Maharashtra) India.

2S.N.D.T. Women’s University, C. U. Shah College of Pharmacy, Sir Vithaldas Thakersay, Santacruz West, Juhu, Mumbai - 400 049, (Maharashtra) India.

4Department of Pharmacognosy, K.L.E. University, College of Pharmacy, Vidyanagar, Hubli - 580031, (Karnataka) India.

*Corresponding Author E-mail: rupalitarange830@gmail.com

 

ABSTRACT:

Background: The threat posed by drug-resistant pathogens has resulted in the increasing momentum in research and development for effective alternative medications. The antioxidant and antifungal properties of phytochemical extracts makes them attractive alternative complementary medicines. Therefore, this study evaluated the phytochemical constituents of Lagenaria siceraria (Molina) Standl. (Cucurbitaceae) leaf extract and their antioxidant and antifungal activities. Aim/Objective: To evaluate the antioxidant and antifungal activity of leaf of L. siceraria. Methods: The leaf of L. siceraria was extracted with hydroalcohol. The extract was subjected to phytochemical evaluation. Antioxidant activity was assayed by the DPPH radical scavenging activity mechanism and the reducing power test. The reducing power assay of extract was carried out with ascorbic acid as a standard reducing agent. All the analysis was made with the use of UV-Visible spectrophotometer. However, in the antifungal activity, the extract was tested by agar well diffusion method by against fungi (Aspergillus niger, Aspergillus flavius and Candida albicans). The leaf extract is compared with standard antifungal drug Amphotericin B. Results: Qualitative phytochemical analysis of hydroalcohol extract of L. siceraria leaf showed a majority of the compound including tannins, alkaloids, flavonoids, saponins and sterol-terpenoids. The hydro-alcohol extracts showed moderate to potent antioxidant activity, among which the leaf extract demonstrated the strongest antioxidant activity with the IC50 value of 26.55 µg/mL. Hydroalcohol extract of leaf possessed antioxidant activity and compared with ascorbic acid and thus could be a potential source of natural antioxidant. In case of antifungal screening, crude-extract of L. siceraria leaf showed notable antifungal activity against tested microorganisms. The leaf extract showed the highest mean zone of inhibition ranging from 9.0-11.0 mm against tested microorganisms, at a concentration of 100 mg/mL. Conclusions: The present finding suggests that the hydroalcohol leaf extract of L. siceraria having antifungal activity, and could be developed as pharmaceutical products.

 

KEYWORDS: L. siceraria, Leaf, Hydroalcohol, Flavonoid, Antioxidant, Antifungal.

 

 


INTRODUCTION:

The cells of living organisms generate free-radicals as a result of pathophysiological and biochemical processes in response to factors such as environmental pollutants, radiation, chemicals, and toxins. This creates an imbalance in the formation and neutralization of prooxidants that subsequently seek stability through electron pairing with biological macromolecules such as proteins, lipids, and DNA, leading to oxidative stress in the physiological system. Furthermore, these effects lead to lipid peroxidation as well as protein or DNA damage or both in human cells. Moreover, the cellular damage consequently lead to aging and several chronic diseases such as cancer, diabetes, and atherosclerosis as well as cardiovascular, inflammatory, and other degenerative diseases in humans, etc.1

 

Fungi have been present for around 1500 million years with more than 1.5 million species out of which only about 300 species are known to cause human infections.2,3 From the mid‑20th century, the incidence of severe systemic fungal infections increased significantly, mainly due to increase in the number of patients with compromised immune system such as acquired immunodeficiency syndrome (AIDS) or postorgan transplantation and chemotherapy. Among all fungal infections, superficial mycoses are the most frequent forms of human infections, affecting more than 20%–25% of the world’s population.4 It is also estimated that 30%–70% of adults are asymptomatic carriers of these pathogens.5

 

Formation of free radicals in body produce oxidative stress then it formed more than 100 diseases like Alzheimer’s, Diabetes-mellitus, Carcinoma, etc.6 90% of all HIV/AIDS patients contract fungal infections and that 10–20% die as a direct consequence of fungal infection.6 Candida spp. and Aspergillus spp. are responsible for more than 80% of all fungal infections in solid organ transplant recipients.6 Here available of synthetic drugs for prevention of oxidation and fungal diseases but it also having a more side effects.

 

According to World Health Organization (WHO), Traditional system of medicine or Herbal medicine is still the mainstay of about 80% of the world population, mainly in the developing countries, for primary health care. This is primarily because of the general belief that herbal drugs are without any side effects besides being cheap and locally available. As per the World Health Organization (WHO), the use of herbal remedies throughout the world exceeds that of the conventional drugs by two to three times.7

 

The ability of certain phytochemical extracts to prevent growth or kill micro-organisms and inhibit or delay the oxidation of other molecules by suppressing the initiation or propagation of oxidizing chain reactions have made them active alternatives in complementary medicine. These naturally occurring antioxidant chemicals have been reported to be composed of phenolic, alkaloids, chlorophyll derivatives, amines and amino acids, carotenoids, ascorbic acid.8 vitamins, flavonoids, alkaloids, carotenoids, terpenoids, polyphenols and phenolic compounds, etc have been reported to exhibit antioxidant, antimicrobial and anticancer activities, etc.9

 

As per literature review here select the medicinal plant Lagenaria siceraria (Molina) Standl. leaf with hydroalcohol extract for helpful in treatment of oxidation and fungal disorder, etc.10

 

The plant, Lagenaria siceraria (Molina) Standl. Belonging to family Cucurbitaceae, known as bottle gourd (Figure 1), is a common fruit vegetable used throughout the India. Its common names include bottle gourd (Eng.); alabu (Sanskrit); lauki or ghia (Hindi); dudhi bhopla (Marathi); dudhi or tumbadi (Gujarati); sorakkai (Tamil); chorakkaurdu (Malayalam); and ghiya (Urdu).10, 11

 

Figure 1: Aerial parts of L. siceraria (Molina) Standl

 

The previous work of L. siceraria (Molina) standl. parts of plant is used as immunosuppressant, antioxidant, hypolipidemic, hyperlipidemic, diuretic, antihepatotoxic, cardio-tonic, cardio protective and nutritive agent, etc. The L. siceraria plant is also reported to have good source of vitamin-B complex, choline along with fair source of vitamin-C and β-carotene, Cucurbitacins, fibres, polyphenol, campesterol and sitosterol, flavones-c glycosides, phytoconstituents, etc. Lagenin, a ribosome inactivating protein (RIP) isolated from the seeds of L. siceraria. possesses immunoprotective, antitumor, anti HIV and antiproliferative properties, etc.12, 13, 14, 15, 16, 17

 

MATERIAL AND METHODS:

Plant Material:

The plant Lagenaria siceraria (Molina) Standl. leaf was collected from local area of Tarangfal (Malshiras) Solapur, Maharashtra state and authenticated by the Pharmacognosist Dr. H. D. Jedage, Associate Professor & Head Department of Pharmacognosy, L. N. B. Chhabada Institute of Pharmacy (Degree), Raigaon, Satara (Maharashtra) India, where a sample (Voucher number: LNBCIOP/P’COG/2018-19/01) was deposited & preserved in herbarium for future reference.

Extraction:

The shade dried leaves of L. siceraria. were extracted by maceration technique. Coarsely grinded leaves were taken in the conical flask; hydroalcohol solvent (water and alcohol mixture, 3:7) was added to wet the powder. Extracts were prepared by adding 100 g of crude drug in 750 ml solvent for 72 h at 25°C. Further sufficient solvent was added and the flask was sonicated using a bath sonicator. Flask was further kept on orbital shaker for the specified time at 80-90 rpm for complete maceration. Macerated slurry was filtered using vacuum. Filtrate, thus obtained was concentrated by evaporation of the solvent to get the dried extract.18

 

Qualitative phytochemical analysis:

The hydroalcohol extract of L. siceraria. leaves were subjected to various qualitative chemical tests to detect the presence of various phytoconstituents in this plant.19,20

 

Determination of total polyphenolic content:

Total polyphenolic content of hydroalcohol extract of L. siceraria. Leaves was estimated using Folin Ciocalteu reagent. Polyphenols were expressed as mg/g equivalent to gallic acid.21, 22,23

 

Antioxidant activity:

2, 2-diphenyl-1-picrylhyrazyl (DPPH) assay:

The free radical scavenging activity of the hydroalcohol extract L. siceraria. were measured in vitro by 2, 2′- diphenyl-1-picrylhydrazyl (DPPH) assay according to the method described earlier.

 

Radical scavenging activity (%) = [(abs control – abs sample)/abs control)] × 100

 

The absorbance was taken at 520 nm against blank as a methanol and IC50 value were also calculated for all samples.24, 25, 26, 27, 28

 

Antifungal activity:

Test microorganisms and growth media:

Aspergillus niger, Aspergillus flavius, Candida albicus, were selected based on their importance. The fungal strains obtained from L. N. B. Chhabada Institute of Pharmacy (Degree), Raigaon, Satara, for evaluating antifungal activity.

 

Preparation of dried filter paper discs:

The Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which are placed in a Petri dish and sterilized in a hot air oven. The loop used for delivering the extracts and standard drug is made of 20 gauge wire and has a diameter of 2 mm. This delivers 0.1 ml (100 µg/ml) of extracts and standard to each disc.

 

Preparation of extract & standard drug:

The hydroalcohol extract of L. siceraria. was prepared 100 mg/ml as a test drug & Standard (Amphotericin B) 100 mg/ml.

 

Agar well disc-diffusion method:

The disk diffusion method is used to evaluate antifungal activity of the hydroalcohol extract of L. siceraria. leaves. 20 ml Meuller Hinton agar medium plates were inverted and allowed to dry and a 37°C incubated for 30 minutes.

 

An overnight culture of fungus i.e. Aspergillus niger, Aspergillus flavius, Candida albicus, etc. (0.5 ml) was spread over the surface of agar plates using sterile glass spreader. Inoculated plates were inverted and incubated at 37°C for further 30 minutes. 100 μl of test drug i.e. hydroalcohol extract of L. siceraria. leaves and Amphotericin B was used as positive control or as a standard drug, was placed onto 6 mm blank antifungal susceptibility discs. The test drug impregnated discs were then placed onto the inoculated surface of the agar plate (Maximum of 5 discs/plate). The agar plates were incubated overnight (24-48 hours) at 37°C and the zone of fungal inhibition were recorded using vernier calipers.29,30,31

 

RESULTS AND DISCUSSION:

The hydroalcohol extract of L. siceraria. leaves were done qualitative chemical tests to detection and confirmation of various phytoconstituents i.e. Carbohydrate, Glycosides, Triterpenoids, Tannins, Phenolic, Saponins, Protein & Amino acids, Flavonoids, etc compounds in this plant (Table 1). Most of these chemical entities are important for the therapeutic usage.

 

Table1: Hydroalcohol extracts of L. siceraria (Molina) Standl.

Sr. No.

Phytoconstituents

Hydroalcohol extract

01

Glycosides

+ ve

02

Triterpenoids

-- ve

03

Tannins

+ ve

04

Alkaloids

-- ve

05

Flavonoids

+ ve

06

Saponins

+ ve

07

Phenolic

+ ve

Where, + ve : Present and -- ve : Absent

 

Polyphenols or flavonoids are the chemical constituents which play important roles in plant and human life. In current study, the total polyphenolic content were determined in the leaf of L. siceraria. The polyphenolic or flavonoid content in the L. siceraria. were found to be 63.09 ± 0.05 mg/g.

 

DPPH is a protonated radical having absorption maxima at 520 nm which decreases with the scavenging of free radical by hydroalcohol extract. Hence, DPPH finds applications in the determination of the antioxidant activity. Hydroalcohol extract of L. siceraria. showed good and powerful antioxidant potential (IC50 value 26.55 μg/ml) and this was compared to standard ascorbic acid.

 

Antifungal activity is important in medical mycology and standard methods have shown remarkable progress in the field of antifungal drug discovery and leads.

 

The antifungal activity of the L. siceraria. leaves hydroalcohol extract was evaluated against three fungal strains i.e. Aspergillus niger, Aspergillus flavius, Candida albicus, etc. The diameter of the zone of inhibition was used as indicator for antifungal activity.

 

The zone inhibition was found of L. siceraria. leaves hydroalcohol extract to Aspergillus niger (12.0-16.0±0.3 mm), Aspergillus flavius (12.0-16.5 ± 0.2 mm), Candida albicus (9.0-11.0±0.2 mm) against tested microorganisms, at a concentration of 100 mg/mL. and the standard antifungal drug shows Amphotericin B (5.0-7.0 ± 0.01 mm) (Figure 2) and (Table 2)

 

 

Figure 2: Antifungal activity of L. siceraria (Molina) Standl.

 

Table 2: Antifungal activity of L. siceraria (Molina) Standl.

S. No.

Group

Dose

Zone inhibition (mm)

1

Control (Solvent)

Q.S. ml

None

2

Standard (Amphotericin B)

100 mg/ml

5.0-7.0 ± 0.01

3

Aspergillus niger

+ HALc

100 mg/ml

12.0-16.0 ± 0.3

4

Aspergillus flavius

+ HALc

100 mg/ml

12.0-16.5 ± 0.2

5

Candida albicus

+ HALc

100 mg/ml

9.0-11.0 ± 0.2

Where, HALc : Hydroaclohol extract of L. siceraria (Molina) Standl.

 

Upon comparison of inhibition diameter recorded, it was observed that the L. siceraria. leaves hydroalcohol extracts shows antifungal and antioxidant activity, but Candida albicus having maximum zone inhibition with good antifungal activity than other two fungal strains, etc.

 

 

 

CONCLUSION:

Hydroalcholic extract of L. siceraria. leaf confirm the antifungal activity but C. albicus is having more activity than A. niger, A. flavius, etc. because presence of strong antioxidant phytoconstituents in hydroalcohol extract L. siceraria leaf i.e. tannins, flavonoids and phenolic compounds, etc.

 

ACKNOWLEDGEMENT:

The authors (Research Scholar) is thankful to Research Guide Dr. H. D. Jedage, Associate Professor & Head Dept. of Pharmacognosy, L. N. B. Chhabada Institute of Pharmacy (Degree), Raigaon, Satara, Maharashtra state, for providing necessary facilities and support to accomplish this research work successfully.

 

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Received on 22.04.2019         Modified on 28.05.2019

Accepted on 21.06.2019       ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2019; 11(3):155-159.

DOI: 10.5958/0975-4385.2019.00026.8