Antioxidant, Antibacterial, and Anticancer Activities of Bitter Gourd Fruit Extracts at Three Different Cultivation Stages

Department of Chemistry, Government College University, Faisalabad 38000, Pakistan Department of Environmental Sciences, Government College University, Faisalabad 38000, Pakistan Department of Biological Sciences and Technology, China Medical University, Taichung 40402, Taiwan Department of Chemistry, COMSAT University Islamabad, Abbottabad Campus, Abbottabad, Pakistan Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt


Introduction
Infections and cancers are two major human diseases across the world. Recently (December 2019), novel corona virus infection  created graveyard threat to all nations over Earth. In a short span (∼3 months) of time about 200 countries victimized with COVID-19. e most advanced states, especially USA and Italy remained fail to cope COVID-19 infection and thousands of deaths were reported in a few days. World Health Organization (WHO) in this scenario declared international emergency. Bacterial infection is another serious threat to humanity as bacterial resistance to antibiotics growing continuously. Cancer is another leading cause of deaths as the oncology field bears limited effective therapeutic strategies.
Antibiotics are the main weapons being used to fight against bacterial infection. e choice of antibiotics, however, decreased due to resistance factor which increased the threat of severe consequences [1]. Infections are estimated to contribute to 20% of all human tumors [2]. It was observed that the patients with bronchogenic carcinomas have active tuberculosis more frequently than the general population. Despite the direct effect of pathogenic organisms which is most probably misstep, the induction of inflammation and production of mutagenic compounds by bacterial metabolism are the two important reasons. Noncardia gastric carcinoma and colon carcinoma are common examples of the bacterium origin cancers [1]. Bacterial infection has attributed about 1.2 million cancer cases per year [3].
Natural products and phytochemicals are gaining the attention in global emergence of bacterial resistance and increasing cases of oxidative stress diseases scenario [4,5]. Phytomedicines carry multiple advantages over infectious and cancerous chemotherapeutic drugs which includes less side effects and nonresistive by bacteria. Plants have long been investigated for new therapeutic agents. Over 80% of the world population and 90% of the African population rely on medicinal plant based therapy [6]. Similarly, pharmaceutical industry during the period of 1981-2002 developed about 61% new natural product based drugs that were highly effective against infectious and cancerous diseases [7]. As reported by WHO, medicinal plants would be the best and cheap source of obtaining the anti-infection and anticancer agents [8].
erefore, screening of the flora for natural products and phytochemicals to suppress and eradicate infection and malignant diseases is one of the primary focuses.
Bitter gourd (BG) is a wild variety of Momordica charantia belonging to family Cucurbitaceae, consumed both as vegetable and folk medicines. Many biologically active compounds have been identified in Cucurbitaceae family including phenolics, steroidal glucosides, alkaloids [9], conjugated linolenic acid isomers [10], lysophosphatidylcholines [11], organosulfur compounds, and cucurbitane-type triterpenoids [12,13]. Certain common diseases such as diabetic mellitus, hypoglycemia, heart disease, HIV (human immunodeficiency virus), cancer, and microbial infections have been investigated to treat with phytochemical fractions and compounds isolated from gourd family [14,15]. e aim of presented study was to investigate the antioxidant, antibacterial, and anticancer activities of BG fruit extracts at three different cultivation stages for taking the maximum advantage of BG in functional food and phytomedicine practice.

Preparation of Extracts.
Fresh bitter gourds were plucked from the fields located around the Faisalabad, Pakistan, at three different stages, that is, immature stage (light green, 8-12-day fruit age, 4-6 cm in length with soft white seeds), mature stage (full green, 16-22-day fruit age, 10-14 cm in length with yellowish white seeds), and ripen stage (full yellow, 30-day-fruit age, 10-14 cm in length with red seeds) ( Figure 1). Fruits were properly washed with distilled water, dried under shade with mechanical air blowing, chopped, and then ground to fine powder having 80-150 mesh size. e phytochemicals from BG fruit powder were extracted in 60%, 80%, and 100% methanol solvent using 1:10 (w/v) powder to solvent ratio at room temperature (RT) using orbital shaker (220 rpm) for 24 h. e extraction mixture was then filtered using filter paper (Whatman No.1). e filtrate was concentrated with rotary evaporator till thick paste at 65°C followed by refrigerating at 4°C for further analysis. Percent yield of the extracts was calculated using the following equation; Weight of solvent free extract(g) × 100 Dried extract weight(g) . (1)

Determination of Total Phenolics. TPC in BG extracts
were investigated with Folin-Ciocalteu reagent (FCR) assay [16]. e assay was carried out by adding 0.5 mL FCR to 50 mg dry extract followed by diluting the mixture with 7.5 mL deionized water (DW), vigorously shaking, and incubation at RT for 10 min. en after adding 1.5 mL sodium carbonate solution (20 %), the mixture was heated for 20 min at 40°C followed by ice-bath cooling. e absorbance of the cooled solution was than measured at 755 nm. e results are presented as mg gallic acid equivalent (GAE) per g dry matter.

Determination of Antioxidant
Potential. Antioxidant potential of bitter gourd was studied from the different solvent extracts using DPPH o scavenging, reducing power, linoleic acid, and ferric reducing power assays.

DPPH Free Radical Scavenging
Activity. DPPH o scavenging assay was performed following the protocol described by Iqbal and coworkers [17]. Briefly, freshly prepared 5 mL methanol solution of DPPH free radical (0.025 g/L) was mixed with 1 mL ethanol solution of extract (25 µg/mL). e contents were then vortexed for 1 min, allowed to stand at RT for 20 min, and then recorded the absorbance at 510 nm. DPPH o scavenging activity was calculated with the following expression.
Percent inhibition of DPPH o � (1 − As/A c ) × 100. A s � absorbance of sample. A c � absorbance of control.

Determination of Reducing Power
Activity. e reducing power of bitter gourd extracts was determined using procedure described by Iqbal and coworkers with slight modification [17]. Methanol extract (0, 10, 20, 30, 40, or 50 mg) was added to 5 mL of sodium-phosphate buffer (pH 6.6). en 5 mL aqueous solution of potassium-ferricyanide (1%) was added and the mixture was heated at 50°C for 20 min. en, 5 mL aqueous solution of trichloroacetic acid (10%) was added to the mixture, centrifuged at 1 krpm for 10 min at 4°C. First supernatant layer was separated and diluted with 5 mL DW, and then 1 mL ferric chloride solution (0.1%) was added for recording absorbance at 700 nm. e experiment was repeated in triplicate and results were expressed mean absorbance.

Percent Inhibition of Linoleic Acid Peroxidation.
Inhibition of linoleic acid peroxidation (LAP) chemical test reported previously was followed to conduct antioxidant activity of methanol extracts [18]. Briefly, to 5 mg dry BG extract subsequent addition of linoleic acid (0.13 mL), absolute ethanol (10 mL), and sodium-phosphate buffer (10 mL, 0.2 M, pH 7.2) was done. e solution mixture was diluted up to 25 mL with distilled water followed by incubation at 37°C for 172 h. e incubated solution mixture (0.2 mL) and 0.2 mL ferrous chloride solution (FeCl 2 ) (20 mM in 3.5 % HCl) were then added into 10 mL ethanol (75%), mixed sequentially and stirred for 3 min and finally recorded the absorbance at 500 nm. A solution without sample used as negative control and with BHT was taken as standard.
e percent inhibition of linoleic acid peroxidation was calculated by the following equation.

Ferric Reducing Antioxidant Power (FRAP).
FRAP assay was carried out as described earlier [19]. Briefly, FRAP reagent was prepared by preparing three different solutions and then mixing them with specific ratio: solution 1: acetate buffer (pH 6; a mixture of sodium acetate (3.1 g) and acetic acid per 20 mL per liter, solution 2: TPTZ (10 mM) in HCL (40 mM), and solution 3: FeCl 3 solution (20 mM). ese three solutions were mixed at 10 : 1:1 (v/v/v) and the mixture was incubated at 37°C for 10 min.
en to the 1 mL of methanol samples (0, 1, 2, 3, 4, or 5 mg/mL), 1.0 mL FRAP reagent was added to test tube, incubated for 30 min at 25°C followed by recording the absorbance at 593 nm. e results were expressed in terms of mean absorbance (directly related to antioxidant potential) of triplicate values ± standard deviation.

Determination of Antibacterial Activities.
Antibacterial activities were evaluated by using disc diffusion method reported in literature [20]. Nutrient agar 13 g/L was suspended homogenously in distilled water and sterilized by autoclaving followed by the addition of 100 μL/100 mL of the inoculums in sterilized culture medium. is medium was then transferred to sterilized Petri plate and allowed to solidify. Small filter paper discs of about 1.5 mm diameter were placed on an aluminum foil and 100 μL of the BGE sample (0.5 mg/µL in DMSO) was dropped on them. ese discs were allowed to dry and then laid flat on growth medium in Petri plates. e Petri plates were then incubated at 37°C for 24 h. At the end of incubation period zone-ofinhibition was measured with the help of zone reader to assess antibacterial potential.
e results were compared with antimicrobial agent rifampicin, taken as standard.

In Vitro Anticancer Activities.
e in vitro anticancer activity was performed with HeLa cancer cell line belonging to human cervical cancer and Madin-Darby bovine kidney (MDBK) cancer cell line belonging to animal cancer following the procedure described by Mosmann [21]. e extracts were diluted at five different concentrations, that is, 0.312 mg/ml, 0.625 mg/mL, 1.25 mg/mL, 2.5 mg/mL, and 5 mg/mL, in labeled wells; already having 2000 cancer cells/ well. DMEM, supplemented with 5% FBS as a cell line growth medium was incubated at 37°C under 5% CO 2 pressure for 48 h. Following the incubation, 20 μL of DMT-DPTB stock solution (5 mg/mL) was added to each well and further incubated for 3 h to form purple formazan crystals. e crystals were then dissolved in DMSO (100 μL/well) to record OD value at 570 nm with reference cisplatin (control) using the following expression: (2)

Statistical Analysis.
e experiments were conducted in three replicates and the generated data was analyzed using CoSTAT V 6.3 software (Cohort software, Berkeley, California, USA).

Extraction Yield and Total Phenolics.
Extraction of airdried BG fruit at three ripening stages was performed in methanol using three different concentrations (60, 80, and 100%). e extraction process was carried out under controlled set of mechanical agitation, humidity, pressure, and temperature conditions. Extraction yield of bitter gourd fruits at different stages with different methanol concentrations is shown in Table 1. e immature fruit contained slightly higher % yield as compared to the mature and ripen fruits. e amount of total PC (TPC) expressed as percent weight of dried extract ranged from 1.91 ± 0.11% to 8.17 ± 0.13% (Table 1). e lowest TPC was noted for immature BG fruit extract in 60% methanol while the highest TPC was found for mature fruit extract in 100% methanol. A significant difference between %yield of extracts and %TPC of immature BG fruit extract indicated that immature fruit contained more nonphenolic compounds which gradually metabolized to secondary metabolites. However, after certain level of maturity variety of other compounds also formed which contribute in biological activities.

Scavenging of DPPH Free Radicals.
e antioxidant activity of BG fruit extract was assessed using DPPH o as it shows neutral behavior with superoxide anion and hydroxyl radicals [22]. Our results showed the highest free radical scavenging potential 78.12 ± 2.64% in case of mature BG fruit extracts in 100% methanol. Both immature and ripen fruit extracts showed less than 60% radical scavenging potential.
e results are shown in Figure 2(a). Previously, antioxidant activity of different parts of BG extracts in water was reported showing leaf, stem, mature fruit, and ripen fruit able to scavenge 83%, 80%, 81%, and 53% DPPH free radical [23]. Rezaeizadeh et al. reported 80% DPPH o scavenging action of methanol extract of whole fruit including seeds at concentration of 500 µg/mL [24]. When compared to BHT, known to be the strong synthetic antioxidant, 79.13 ± 2.43% free radical scavenging activity was found which was slightly greater than 100% methanol extract of mature BG fruit (78.12 ± 2.64%). Not only did the higher value of DPPH o scavenging of fruit extract attribute to PC but also other compounds also effect the antioxidant activity which is commonly known as synergism.

Percent Inhibition of Linoleic Acid Peroxidation (LAP).
Oxidation of lipids, carried out by ROS, paved the way to pathogens to initiate infectious diseases. Generally, ROS attack polyunsaturated fatty acid chains of cell membrane which initiates self-propagated chain reaction. Results of inhibition potential of LAP by different stages of BG fruit are shown in Figure 2(b). Extraction in 60% methanol solution showed poor inhibition of LAP (21%-31%), while 80% methanol solution extracted good level of antioxidants from BG fruit. e promising extraction was carried out by 100% methanol solvent. is was reflected by favorable inhibition of LAP (34% to 57%). Inhibition of linoleic acid peroxidation largely shown by mature stage of BG fruit (56.78 ± 1.64%) when extracted in 100% methanol solvent-other stages, that is, immature stage, showed inhibition of LAP showed 34.21 ± 3.52% while ripen stage showed 49.56 ± 1.23%. e standard antioxidant compound (BHT) showed 72.45 ± 2.66% inhibition of LAP which is more than crude fruit extract. Butylated hydroxyl toluene is the most widely used synthetic antioxidant but it has been restricted to use as an antioxidant because of its carcinogenic potential [24,25].

Reducing Power Activity.
Published researches have demonstrated that the electron donating potential of naturally extracted compounds is associated mainly with its reducing power [26]. Figures 3(a)-3(c) demonstrate the reducing power of different BG fruit extracts. In general results showed concentration dependent reducing power ability which directly related to absorbance. e highest reducing power (absorbance � 1.05) was noted with 100% methanol extract sample followed by mature BG fruit extract sample (absorbance � 0.97) extracted in 80% methanol solution. Ripen fruit extracts also showed satisfactory reducing ability; extraction in 100% methanol showed absorbance 0.69 while immature fruit showed poor reductive ability. e reducing power of standard ascorbic acid showed 1.8 absorbance at 700 nm which was higher than all extracts. e assay is based on the ability of natural extract to change the yellow color of the test solution to various shades of green and blue depending on reduction of Fe3 + to Fe2 + which is then compared to reduction ability of ascorbic acid, which is known as best reducing agent.

Ferric Reducing Antioxidant Power.
e FRAP assay is commonly used for the antioxidant studies of plant extracts which is based on the electron-transfer reaction. e mechanism is carried out as the colorless [Fe3 + ]-TPTZ accepts an electron from antioxidants to generate the colored [Fe2 + ]-TPTZ adduct. e absorbance of visible light directly related to the electron donating potential of antioxidants. e ferric reducing antioxidant power of immature, mature, and ripen BG fruit extracts in 60%, 80%, and 100% was measured and the results are shown in Figures 3(d)-3(f ). Generally, the FRAP activity of all samples was found concentration dependent.
e maximum absorbance of sample solutions was noted with 5 mg extract at 593 nm. Mature BG fruit extract in 100% methanol showed 0.73 absorbance whereas 80% and 60% methanol extract showed absorbance of 0.67 and 0.53, respectively. Immature and ripen fruit extracts, however, showed comparatively low absorbance. It was also noted that pure methanol extract appeared to be more active than 80% and 60% methanol extracts.   3.6. Antimicrobial Activity. Variety of Momordica species have been reported to bear good antibacterial activities against both Gram-positive and Gram-negative bacteria [27]. It has been suggested that phenolics containing polar isopropyl functionality are responsible for antibacterial activity of natural extracts [28]. e results indicate mature and immature BG fruit extracts are more antibacterial as compared to ripen fruit extracts. e extracts were found promisingly active against both Gram-positive (S. aureus and B. subtilis) and Gram-negative bacteria (E. coli and P. multocida). e extracts in 60% methanol were also found less active against bacterial inhibition irrespective of the maturity level. e differences in inhibition activities were found significant at P < 0.001 as shown in Table 2. According to the previously reported data, it has been suggested that phenolic compounds damage bacterial membrane, for example, S. aureus on treating with BHA (bone-like hydroxyapatite), releases nucleotide into culture medium [29], whereas exposure to P. fragi and P. fluorescens increased the leakage of protein material [30]. In the present study, the yield of BG fruit extract with 60% methanol is comparable with 80% and 100% methanol extracts but it is not warranted that their bacteriostatic activity will also be similar. However, by increasing the methanol concentration in extracted solvent the antibacterial activity also increased [29,31]. Previously, Ozusaglam and Karakoca investigated the antibacterial activity of unripen and ripen ethanol extracts of BG seeds and fruits and found unripen BG fruit extracts are more active against microorganisms [32]. Alkaloids, glycosides, volatile oils, or tannins in addition to phenolics and flavonoids are known to present in BG fruits which largely attribute to antimicrobial activities [33].
ese are best evaluated for antibacterial activities when extraction was performed with methanol solvent [33][34][35]. It is worth taking to mention that combined antibacterial effect of all compounds present in fruit extracts not only hacks the microbial activity and growth but also makes microbes unable to produce resistance mechanism. IC 50 values (calculated using S. aureus bugs) indicated immature BG fruit extract in 80% methanol showed lowest IC 50 (513 µg/mL) while in same solvent ripen BG fruit extract showed IC 50 789 µg/mL which is greater than any other extract as shown in Table 3. Our study clearly showed two main facts: immature and mature fruit extracts in concentrated methanol exhibited great antibacterial activity as compared to more aqueous and ripen fruit extracts.

Antiproliferative Activity.
A multitude of plants and their different parts particularly fruits have been extensively investigated for the therapy of cancer [36]. Proliferation of cell mainly associated with malignant cells. Cancer is the most leading cause of mortality throughout the world beyond the discrimination of developing and developed countries. Chemotherapy, surgery, and radiotherapy are first line options to treat cancer. Chemotherapy mechanism mainly involves targeting the infected cells either to destroy the malignant cells or to stop the proliferation of cells by interfering into one or more different steps of cell division mechanisms [37]. It has been investigated that BG plant extracts showed broad-spectrum antitumor and anticancer activities and have great ability to slow down the cell proliferation rate. e results revealed that mature BG fruit extract in 100% methanol showed 72% MDBK cancer cell death followed by extracts in 80% methanol (40% inhibition) of ripen BG fruit. e 60% methanol extracts also showed high depletion of MDBK cancer cells. However, the cell line depletion activity using HeLa cancer cell line was recorded comparatively low as shown in Figure 4.
In case of MDBK cancer cell line, the lowest IC 50 value (3.56 mg/mL) was calculated for 100% methanol extract of ripen BG followed by 100% methanol extract of mature BG (7.95 mg/mL), 80% methanol extract of ripen fruit (9.02 mg/ mL), and 80% methanol extract of mature fruit (9.08 mg/ mL) ( Table 4). In contrast to MDBK cancer cell line, HeLa cancer cell line showed slightly less sensitivity to BG fruit extracts. e results showed that at 5 mg/mL concentration, the 100% methanol extract of ripen fruit showed maximum inhibition of cancer cell growth (65.80 ± 3.10%), followed by 60% methanol extract (46.64 ± 3.74%) and 80% methanol extract (41.60 ± 4.9% at 2.5 mg/mL). Other methanol extracts (in 80% and 60% methanol) also showed similar activities that were observed in case of 100% methanol extract but significantly less. e IC 50 value was also calculated that was greater than MDBK cell line (Table 4). e viability of the MDBK cancer cells treated with 60%, 80%, and 100% methanol ripen BG fruit extracts was imaged with trinocular microscope as shown in Figure 5. e control drug (cisplatin) showed maximum cell death (82.45 ± 2.31%) followed by 80% and 100% methanol extracts of BG.
Different groups have reported that BG extracts play active role against number of common cancers such as

Mature Ripen
LSD 5% MDBK cancer cell line 100% ME 80% ME 60% ME LSD 5% HeLa cancer cell line Similar signs show no significant difference while nonsimilar signs show significant difference at level P < 0.001 100% ME 80% ME 60% ME lymphoma [38], skin tumour [39], breast cancer [40], lymphoid leukemia [41], prostatic cancer [38,42], melanoma, and choriocarcinoma [27]. Furthermore, it has also been reported that aqueous extract of BG more actively inhibits the growth of prostatic adenocarcinoma [43], breast cancer, and human bladder carcinomas. Various compounds such as MAP30 (Momordica anti-HIV protein), momordin, and alpha-momorcharin isolated from BG extracts have shown anticancer activities. MAP30, a protein based molecule of 30 kDa mass in addition to topological inactivation of HIV-LTR-DNA, also induce cell damaging of breast cancer cells xenograft into mice [44]. Our results are also in good agreement with reported studies.

Conclusion and Future Perspective
Metabolic products of plants play paramount role in the eradication or slowdown the number of disease processes. BG is well known for its incredible medicinal activities against numerous diseases, especially diabetes mellitus. In this study, our investigations to see the effect of three different ripening stages of air-dried BG extracts on antioxidant, antibacterial, and anticancer activities reveal promising results. e mature BG fruit extract in absolute methanol showed strong antioxidant activity than immature and ripen fruit extract, while immature and mature fruit were found to show promising antibacterial activity and ripen fruit extract in 100% methanol was found to have (c) (d) Figure 5: e effect of the ripen BG extracts (incubation with 5 mg/mL extract) on the viability of the MDBK cancer cells. Most of the cancer cells remained healthy and viable when treated with 60% methanol extract (a), treating with 80% ripen BG fruit extract showed cells depletion effect but not prominently (b), treating with ripen BG extract in 100% methanol showed strong deformation of cell structure (c), and control methotrexate anticancer drug showed very strong cell death effect (d). anticancer activity. On the bases of the results, we can conclude that the immature and ripen fruits that commonly wasted at the end of BG cultivation season could be collected and air-dried for obtaining the extracts as an antibacterial and anticancer activities by including in functional food as an ingredient or as an Ayurveda medicine.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors declare that no conflicts of interest exist.