Bioactivity of Ethanolic Extract of Borassus flabellifer Sprout Peel and Its Phytochemical Analysis: An In vitro Study

Abstract

Palmyra palm sprout skin was extracted using ethanol to test its antibacterial activity against isolated urinary tract infection (UTI) pathogenic bacteria. The extract demonstrated excellent inhibitory activity at the concentration of 500 µl/well. It was observed that all the isolated UTI-causing bacteria were highly resistant to selected commercially available antibiotics. The ethanol extract of the palmyra palm sprout skin showed excellent antioxidant, anti-inflammatory, antidiabetic, and antiproliferative activities. Before boiling the Panam kilangu, the sprout skin is usually peeled from it and thrown away as it is considered of no use. However, our study demonstrates a waste-to-wealth based alternative use for the palmyra palm sprout skin. Based on studies, these by-products could be used as a source of valuable bioactive compounds. It showed excellent inhibitory activity at the concentration of 500 µg/well against UTI bacteria. It also showed to possess various secondary metabolites, such as flavonoids, saponins, etc. Gas chromatography–mass spectrometer analysis revealed the presence of various bioactive metabolites.

INTRODUCTION

When tested with a positive dipstick or urinalysis, it is evident that known pathogenic bacteria are present more than 10,000 cfu/ml, being the cause of urinary tract infections (UTIs), according to studies.[1] Murray et al. stated that worldwide, 150 million people are infected every year by UTIs.[2] Wilson and Gaido reported that universally indicative/characteristic UTIs result in about 7 million visits to outpatient clinics, 1 million visits to emergency departments, and 100,000 hospitalizations annually.[3] Among this, studies show that women are more seriously and repetitively prone to UTIs than men.[4] In human beings, it remains the most common bacterial infection and also commonly occurring nosocomial infection.[5]

Studies reveal that pathogenic bacteria, such as Escherichia coli, Klebsiella, Enterococcus, and Staphylococcus are responsible for UTIs in almost all cases.[6] UPEC or uropathogenic E. coli is known to be the most common bacterial pathogen responsible for causing around 70%–90% of all the urinary infections.[7] Audu and Kudi stated that a high incidence of S. aureus in women and men could be due to the propinquity between the genital tracts and the urethra and anus, which possibly enable to auto transmission.[8] It is becoming more challenging by the day to treat these infections, as the infection-causing micro-organisms are evolving to become resistant to most of the currently used antibiotic treatments. One of the reasons could be the aberrant use of antibiotics in clinical practice.[9] Despite this, antibiotics are still the most commonly used standard treatment methodology for UTIs. However, this could not be beneficial for upcoming years or generations, as the treatment may become almost ineffective. This shows the significant necessity for the immediate alternative solution to identify, develop, and bring to market a new realm of antimicrobial agents with broad-spectrum activity, which would act as an enhanced and reliable alternative therapy for UTIs.

Borassus flabellifer, which is commonly known as palmyra palm, gets its name from the Portuguese word “Palmeira” as the leaves resemble the palm of hands.[10] This belongs to the Arecacean family, and is widely common in Asian countries. Palm is valued for its beneficial properties in almost all of its parts, playing major roles in food, beverage, fiber, and timber industries.

In India, palms are produced nearly 122 million per year.[11] Palmyra is celebrated and honoured to be the state tree of Tamil Nadu since 1978, owing to its widest distribution across the state, which serves various ecological, medicinal, economical, and sociological benefits to the society.[10] According to Gummadi et al., Palmyra products are known to have anti-inflammatory, antiarthritic, cytotoxic, antibacterial, analgesic, antipyretic, hypoglycaemic, and antioxidant properties.[12]

Palmyra sprouts are edible food sources that are commonly preferred in the winter season.[13] The seeds have the ability to grow naturally and are also being sold commercially by local people. The sprout is locally called Gaygulu in Telugu and Panang kizhangu in Tamil. Selling sprouts has shown to be a good commercial business for local people during the peak winter season.[14] The seedlings of the palmyra palm are eaten either freshly or after being dried in the sun, or even after being cooked or as such, depending on the consumer’s taste. The stem of the sprout is very fibrous and nutritious.[15] However, palmyra palm sprouts and skin/peel are usually wasted, or used as a fuel source locally. From these waste products, our study aims to prepare an ethanol extract of palmyra palm sprout peel and to evaluate the antimicrobial activity of ethanol extract of palmyra palm sprout peel (B. flabellifer) against UTI pathogens.

MATERIALS AND METHODS

Sample collection

B. flabellifer sprouts were purchased from Kulathur, Tuticorin district, Tamil Nadu, India. The collected palmyra palm sprout was peeled and rinsed well using distilled water and was dried under shade. Followed by, the dry palm sprout peels were powdered using a grinder and preserved in air-tight containers safely until further work.

Preparation of extracts

100 ml of ethanol was added to 12 g of palmyra palm sprout skin powder in a conical flask. The flask was covered with a cotton plug and placed in a shaker for 3 days in 150 rpm. After that, the ethanol extract of the palmyra palm sprout skin was poured into petri plates for evaporation. Then, the residue was dissolved in dimethyl sulfoxide (DMSO). The ethanol extract of the palmyra palm sprout skin was then kept for preservation in sterile containers until further use.

Collection of urinary tract infection pathogens and determination of antibiotic resistance

Organisms such as Staphylococcus aureus, Klebsiella pneumoniae, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, and Proteus mirabilis were collected from KG Nursing Home, Tuticorin, Tamil Nadu, India. Antibiotic susceptibility was determined using standard antibiotic discs such as streptomycin (10 mcg/disc), chloramphenicol (10 mcg/disc), ampicillin (10 mcg/disc), levofloxain (10 mcg/disc) against UTI bacteria by agar disc diffusion method on Muller–Hinton agar. The pathogenic bacteria, which were isolated, were cultured on the Muller–Hinton agar medium, along with commercially available standard antibiotic discs on the agar plate surface. The plates were kept for incubation under aerobic conditions, at 37°C for 24 h. Followed by, the diameters of zones of inhibition were measured in mm.

Antibacterial activity of ethanol extract of palymra palm sprout skin against urinary tract infection bacteria

The ethanol extract of the palmyra palm sprout skin was analyzed for its antibacterial activity against the UTI-causing bacteria by performing the agar well diffusion method on Muller–Hinton medium. Seed culture preparation was done by adding 1 ml of the log culture of UTI bacteria into 5 ml of nutrient broth. After 24 h of incubation, the organisms were swabbed all over the surface of agar plates, before which the wells were cut at the size of 5 mm onto the agar medium using a sterile well cutter. After that, different concentrations (100, 200, 300, 400 μ/well) of 20 μl ethanol extracts mixed in DMSO were poured into the respective wells. Plates were kept for incubation at 37°C for 24 h, after which zones of inhibition surrounding the wells were measured in millimeters (mm) and recorded.

Determination of minimal inhibitory concentration and minimum bactericidal oncentration

The broth dilution method was used to determine the minimal inhibitory concentration (MIC) or minimum inhibitory concentration of the ethanol extract of palmyra palm sprout skin. Different concentrations of 0.5 ml of ethanol extract of palmyra palm sprout sample (350, 400, 450, 500, 600, 700, and 800 mg/ml) were mixed with 1 ml of nutrient broth, and a loopful culture of the respective test organisms was added. Then, test tubes were kept for incubation at 37°C for 24 h, followed by which microbial growth was determined by the presence of turbidity (OD value).

The minimum bactericidal concentration (MBC) of the ethanol extract of the palmyra palm sprout skin was determined according to the MIC values. The particular concentration showing low OD value (turbidity) (0.1 ml) was spread on nutrient agar medium, and the plates were incubated at 37°C for 24 h. The concentration where no visible growth was observed was considered the MBC value.

Isolation of fungi

Fungal samples were isolated from a source of spoiled vegetables at home. Rose Bengal chloramphenicol agar, a very commonly used fungal media was prepared and sterilized, and poured on sterile Petri dishes. After inoculating the collected samples in sterile plates, they were kept for incubation at 25°C ± 2°C for 3 days.

Antifungal susceptibility

Antifungal activity of standard antibiotics against the isolated fungi was analyzed by the agar disc diffusion method. Two commercially available fungal antibiotics (fluconazole and nystatin) were selected for this study. Rose Bengal agar plates were prepared and allowed to solidify. The organisms were swabbed on the surface of agar plates, after which, the standard antibiotic discs were placed on the surface of the agar plates. The plates were then incubated at 37°C for 48 h. After incubation, the diameters of zones of inhibition were measured around the well in mm.

Antifungal activity of ethanol extract

Antifungal activity of ethanol extract of palmyra palm sprout skin was performed against an isolated fungus by the agar well diffusion method. Rose Bengal chloramphenicol agar medium was used for this study.

Determination of minimum fungicidal concentration

The minimum fungicidal concentration (MFC) could be defined as the lowest concentration of ethanol extract of palmyra palm sprout skin required to kill fungi over a fixed or extended period, for instance, 48 h, under specific conditions. From the broth dilution of the MIC tests, one must subculture the samples into agar plates without the test fungi to determine the MFC. Each tube containing 1 ml of potato broth at different concentrations of ethanol extract of Palmyra palm sprout skin (10, 15, 25, 50, 60 μl/ml), was inoculated with an equal amount of (0.1 mm) agar plug of respective fungi. The plates were kept for incubation under aerobic conditions at 35°C for a period of 48 h, after which the results were observed. The plate with the maximum concentration has no fungal formation, whereas the MIC test, which helps indicate the lowest level of extract that greatly inhibits fungal growth, was selected for the MFC Value. Those samples were inoculated into the Rose Bengal Chloramphenicol agar medium, and plates were kept for incubation at 28°C for 48 h. The MFC value is the concentration where no visible growth was observed.

Microscopic observation of fungicidal activity

Experimental fungi such as Aspergillus niger are grown on clean glass slide using the slide culture technique. After 92 h, fungal growth observed on the slide was taken for this study. One slide was used as control. The slide was stained with fungal stain Lactophenol cotton blue and its morphology was observed under microscope. Another slide was an experimental one. The fungal growth observed slide was treated with 0.5 ml of ethanol extract of the palmyra palm sprout skin. This slide was kept for incubation for 24 h. After that, it was stained with lactophenol cotton blue, and the mycelial morphology was observed under the microscope.

Antidiabetic activity

To understand the antidiabetic activity expressed by the extracted palm sprout skin, an α-amylase inhibition assay was performed using the 3,5-dinitrosalicylic acid (DNSA) method.[16] The ethanol extract of palmyra palm sprout skin powder was dissolved in 10% DMSO and dissolved in 0.02 M buffer ([NaHPO4/NaH2PO4] and 0.006 M NaCl (0.006 M) adjusted to the final pH of 6.9) to yield different concentrations in the range of 10–1000 μg/ml. 200 μl of the α-amylase solution in 2 units/ml concentration was added to 200 μl of the ethanol extract and was incubated for 10 min at 30°C. Followed by, 200 μl of the starch solution (1% in water [w/v]) was added to each of the tubes and kept for incubation for 3 min. 200 μl of DNSA reagent was added in order to terminate the reaction, and this solution was subjected to boiling at 85°C–90°C for 10 min in water bath. Once it reached room temperature, it was diluted using about 5 ml of distilled water, and the absorbance was recorded at 540 nm using a ultraviolet-Visible spectrophotometer.

The % α-amylase inhibition of the ethanol extract of the palmyra palm sprout skin was calculated by the formula:

% Inhibition = ([Absorbance Control-Absorbance Test]/Absorbance control) ×100

Protein denaturation inhibition activity

The reaction mixture with 0.45 ml of BSA or bovine serum albumin (5% aqueous solution) and 0.1 ml of the sample at 1 mg/ml concentration was prepared. The samples were taken in a test tube and kept for incubation at 60°C for 30 min. The samples were allowed to cool, and to each tube, 2.5 ml of Bradford reagent was added. The absorbance was measured at 660 nm using a spectrophotometer. 0.05 ml distilled water was used as control.

The percentage inhibition of protein denaturation was calculated by the following formula:

% Inhibition = (Absorbance Test/Absorbance control) × 100

Antioxidant activity

Antioxidant activity of ethanol extract from palmyra palm sprout skin powder was done by the phosphomolybdenum assay.[17] 0.1 ml of the sample was taken in different concentrations (25–400 μg/mL) in tubes and they were all treated with 1 mL of the solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. In a water bath, the tubes were incubated at 95°C for 90 min. Once the samples cooled down to ambient temperature, the absorbance was measured at 765 nm. Ascorbic acid was used as a positive control. Antioxidant capacity was estimated using the following equation:

Antioxidant activity % = ([Absorbance control − Absorbance sample]/Absorbance Control) ×100

Antiproliferative activity

Cell culture maintenance: MDA-MB-231, breast cancer cell line, was purchased from the cell repository of NCCS, National Centre for Cell Sciences, Pune, India. For the culture, DMEM or Dulbecco`s Modified Eagle Media was used, supplemented with 10% FBS or Fetal Bovine Serum. Penicillin and streptomycin were added to the medium at a concentration of (100U/ml) and (100 μg/ml), respectively, to prevent bacterial contamination. The cell lines were maintained in a 5% CO₂ incubator at 37°C until confluency was observed.

MTT assay: the cytotoxicity of the sample on MDA-MB-231 cells was determined by the method of Mosmann.[18] MTT (Promega, USA) or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to measure the antiproliferative activity of ethanol extract from palmyra palm sprout skin powder. This assay is used to measure the cell viability and the metabolic activity in vitro.[19]

The confluent cells were washed well and seeded in 96-well plates at a concentration of 17,000 cells/well (in 200 μl of growth medium). After a 24-h incubation period, a partial monolayer was observed. This old medium was discarded and replaced with 200 μl of fresh medium containing the plant extract dissolved in DMSO. 100 μl of media was removed and 15 μl of the MTT solution was added. After keeping the plate for incubation for 4 h, the formation of blue crystals was observed. 100 μl of the stop solution was added and incubated for 1 h. The absorbance was measured at 550 nm with a microplate reader (Das, Italy). Control groups received the same amount of DMSO (0.1%). Untreated cells were used as a negative control, whereas cells treated with vincristine sulfate were used as a positive control at the following concentrations - 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100nM.

Qualitative analysis of phytochemicals

The phytoconstituents present in the ethanol extracts of the palmyra palm sprout skin were determined qualitatively with the help of the standard protocols.[20]

Gas chromatography–mass spectrometer analysis

Using a gas chromatography–mass spectrometer (GC-MS) system, 2 μl of the palmyra palm sprout skin ethanol extract was injected into the system’s capillary column (30 m), with 0.25 mm film thickness (30 m 60.2 mm id. coated with UCON HB 2000). The temperature settings were adjusted as follows: initial oven temperature −40°C for 4 min, increased to 25°C at 158°C/min, and then held at 25°C for 10 min. GC-MS was run under computer control at 70 eV. Ammonia was used as the reagent gas to perform chemical ionization at 95 eV. Upon GC-MS analysis, the peak of the dichloromethane solvent was observed at 2.4 min. The unknown compounds in the sample were identified by probability-based matching using systems such as WILEY Registry™95, NIST05, and NIST05s. The compounds present in the palmyra palm sprout skin sample were identified from the GC-MS compounds by comparing the obtained spectrum with the MS data library.

RESULTS

Antibiotic susceptibility test

Table 1 demonstrates the effect of commercially available antibiotics against UTI bacteria, all of which showed high resistance except for Levofloxacin. Levofloxacin showed a negligible amount of sensitivity against all the isolated UTI bacteria. In our study, isolated UTI bacteria were highly resistant to commercial antibiotics.

Table 1.

Antibiotic susceptibility test of urinary tract infection pathogens

Test organisms	Streptomycin	Ampicillin	Chloramphenicol	Levofloxacin
Staphylococcus aureus	Resistance	Resistance	Resistance	Resistance
Klebsiella pneumonia	Resistance	Resistance	Resistance	2 mm
Escherichia coli	Resistance	Resistance	Resistance	3 mm
Bacillus subtilis	Resistance	Resistance	Resistance	5 mm
Pseudomonas aeruginosa	Resistance	Resistance	Resistance	Resistance
Proteus mirabilis	Resistance	Resistance	Resistance	Resistance

The antibacterial activity of ethanol extract against urinary tract infection bacteria

The antibacterial activity of the ethanol extract of palmyra sprout peel was evaluated against the UTI pathogens, which showed excellent activity at the concentration of 500 μl/well. The maximum antibacterial activity of ethanol extract of palmyra sprout skin was shown against B. subtilis and P. aeruginosa (12 mm) and followed by E. coli and P. mirabilis at 11 mm. K. pneumoniae was at 10 mm and S. aureus at 8 mm. The antibacterial activity of the ethanol extract against UTI bacteria is shown in Figure 1.

Figure 1.

Antibacterial activity against UTI bacteria

Minimum inhibitory concentration and minimum bactericidal concentration

The values of minimum inhibitory concentration and minimum bactericidal concentration of ethanol extract of palmyra palm sprout peel (MIC) are shown in Table 2.

Table 2.

Minimum inhibitory concentration and minimum bactericidal concentration

UTI pathogens	MIC value	MBC value
Staphylococcus aureus	700	800
Klebsiella pneumoniae	700	800
Escherichia coli	700	800
Bacillus subtilis	700	800
Pseudomonas aeruginosa	700	800
Proteus mirabilis	700	800

UTI=Urinary tract infection, MIC=Minimal inhibitory concentration, MBC=Minimum bactericidal concentration

Susceptibility of commercial antibiotics against isolated fungi

Fungi isolated from spoiled vegetables were identified as A. niger, Aspergillus flavus, and Aspergillus brasiliensis based on the conidial arrangements by Lactophenol cotton blue staining (result not shown here). The result of susceptibility of commercial antibiotics against isolated fungi is shown in Table 3, where two organisms (A. flavus and A. niger) were resistant to fluconazole, and one organism (A. niger) was resistant to nystatin. The ethanol extract of palmyra palm sprout peel showed the highest inhibitory effects against all the three isolated fungal strains, with the highest zone of inhibition observed in (7 mm) diameter against Aspergillus brasiliensis at the concentration of 300 µl/well. The zone of inhibition observed against A. niger was 6 mm. The minimum zone of inhibition was observed in A. flavus (1 mm). The antifungal activities of the ethanol extract of the palmyra palm sprout peel against fungi isolated from spoiled vegetables are shown in Table 4. The value of minimum inhibitory and MFC of ethanol extract from palmyra palm sprout skin (MIC and MFC) are shown in Table 5.

Table 3.

Susceptibility of commercial antibiotics against isolated fungi

Fungi	Fluconazole	Nystatin
Aspergillus flavus	Resistance	0.2 mm
Aspergillus brasiliensis	15 mm	0.3 mm
Aspergillus niger	Resistance	Resistance

Table 4.

Antifungal activity of ethanol extract of palmyra palm sprout skin

Fungi	Zone of inhibition (mm)
	100 µL/well	200 µg/well	300 µg/well
Aspergillus brasiliensis	4	5	7
Aspergillus niger	3	5	6
Aspergillus flavus	1	No growth	No growth

Table 5.

Minimum inhibitory and minimum fungicidal concentration

Fungi	MIC (µL/mL)	MFC
Aspergillus flavus	700	800
Aspergillus brasiliensis	700	800
Aspergillus niger	700	800

MIC=Minimal inhibitory concentration, MFC=Minimum fungicidal concentration

Anti-diabetic activity (α amylase inhibitory activity)

The α-amylase inhibitory activity of the ethanol extract of the palmyra palm sprout skin was expressed as percent inhibition, which was observed to be 53.64. The results indicated the presence of antidiabetic activity of the ethanol extract of the palmyra palm sprout skin [Table 6].

Table 6.

Alpha amylase inhibitory activity

Sample	Absorbance at 510 nm	Percentage inhibition
Control	1.510	-
PP (ethanol extract of palmyra palm sprout skin)	0.700	53.64

PP=Palmyra palm

Protein denaturation

The protein denaturation of ethanol extract of the palmyra palm sprout skin was expressed as percentage inhibition, which was observed to be 80.64. This result showed that the extract had excellent anti-inflammatory activity. Table 7 shows the percentage inhibition of protein denaturation of the ethanol extract of the palmyra palm sprout skin.

Table 7.

Percentage inhibition of protein denaturation

Sample (mg/mL)	Absorbance at 660 nm	Percentage inhibition
Control	1.240	-
Extract	1.000	80.64

Antioxidant activity

The ethanol extracts of B. flabellifer sprout peel/skin have presented antioxidant activity of 55.88% at a concentration of 1000 µg/ml [Table 8].

Table 8.

Antioxidant activity

Sample	Concentration (µg/mL)	OD at 695 nm	Percentage antioxidant activity
Ascorbic acid	1000	1.700	-
PP	1000	0.950	55.88

PP=Palmyra palm, OD=Optical density

Anti-proliferative activity against cancer cells

In the present study, ethanol extract of ethanol from the palmyra palm sprout peel showed excellent antiproliferative activity against breast cancer cells, as demonstrated in Table 9.

Table 9.

Cell inhibition of breast cancer cells

Control	100 µg	200 µg	300 µg	400 µg	500 µg
0	3.33	10.42	18.33	45.00	59.58
0	6.67	13.33	22.08	49.58	63.33
0	8.75	16.25	28.25	57.92	68.75

Phytochemical analysis

In the phytochemical analysis of ethanol extract of palmyra palm sprout peel, the presence of alkaloids, terpenoids, steroids, flavonoids, saponins, and the absence of tannins was observed, which is shown in Table 10.

Table 10.

Phytochemical analysis

Phytochemical	Result
Saponins	+
Tannins	−
Flavonoids	+
Terpenoids	+
Steroids	+
Alkaloids	+

+=Presence, −=Absence

Gas chromatography–mass spectrometer analysis

The results of GC-MS analysis of ethanol extract from palmyra palm sprout skin show the presence of various bioactive compounds such as Benzoic acid, 2-hydroxy-methyl ester, linoleyl acetate, 9-octadecenoic acid (Z)-, oxiranylmethyl ester, tetrapentacontane, 9-octadecenoic acid (Z)-, oxiranylmetyl ester, butyl 9,12-octadecadienoate, etc. The results are demonstrated in Table 11 and Figure 2.

Table 11.

Peak report of gas chromatography – mass spectrometer

Peak number	Reaction time	Area	Area (%)	Name
1	9.841	2,600,040	9.53	Benzoic acid, 2-hydroxy-, methyl
2	22	18,672,867	68.44	Diethyl Phtalate
3	528	1,236,135	4.53	Linoleyl acetate
4	40.194	900,925	33.30	9-Octadecenoic acid (Z)-, oxiranylmethyl ester
5	40.289	307,847	1.13	Tetrapentacontane
6	40.745	218,214	0.80	9-Octadecenoic acid (Z)-, oxiranylmethyl ester
7	43.528	224,787	0.82	Butyl 9, 12 -octadecadienoate
8	43.585	318,999	1.17	9-Octadecenoic acid (Z)-, oxiranylmethyl ester
9	45.761	276,546	1.01	Tetrapentacontane
10	45.565	386,839	1.42	Gamma, -Sitosterol
11	45.621	236,677	0.87	2, 3-Dihydroxypropyl cis-13-docosenoate
12	46.356	1,903,643	6.98	Glycidyl (Z)-9-nonadecenoate
		27,283,519	100.00

Figure 2.

GC – MS analysis

DISCUSSION

Resistance to antimicrobials is an increasing worldwide problem, especially with UTI pathogens.[21] Flores-Mireles et al. reported that high recurrence rates and increasing antimicrobial resistance among uropathogens threaten to greatly increase the economic burden of these infections.[6] The rise of high rates of antibiotic-resistant organisms causing UTIs has become a public health concern worldwide due to extensive misuse of antibiotics in practice.[22] As a result of this, other strategies have been pursued and used in the treatment of UTIs and amongst them is the use of medicinal plants.[23] Our study focused on analyzing the antibacterial, antioxidant, anti-inflammatory, antidiabetic, and antiproliferative activities expressed by the ethanol extract of the palmyra palm sprout peel. UTI pathogenic bacterial strains showed resistance to commercial antibiotics, whereas our ethanolic extract presented excellent antibacterial properties against the strains. Similar results have been published by Nain et al. and Kuete et al., in which it was shown that the ethanol leaf extract B. flabellifer exhibited a potent inhibitory effect, suggesting the presence of antibacterial phytochemicals such as flavonoids, alkaloids, steroids, terpenoids, and saponins, phenolic, and polyphenolic compounds.[24,25] Several reports stated that different parts of the B. flabellifer Linn. plant comprise biological activities such as antibacterial activity of the fruits, and wound healing.[26] Duddukuri et al. reported that the seed coat aqueous crude extract of B. flabellifer Linn. possessed antimicrobial activity.[27] Information on bioactivity is very less so far. Recent studies have revealed that resistance against azole drugs is becoming common.[28] The ethanol extract of the palmyra palm sprout peel showed the highest inhibitory effects against all the three isolated fungal strains that were used in the study.

Chandran et al. reported that the ethanol extracts of B. flabellifer Linn. leaves have scavenged free radicals in a dose-dependent manner.[29] Plant metabolites such as tannins, catechins, flavonoids, and other phenolics express extraordinary antioxidant properties. Guntupalli et al. stated that secondary metabolites of plants possess strong antioxidative property.[30] Similarly, the ethanol extracts of B. flabellifer sprout peel/skin also showed antioxidant activity of 55.88% [Table 8]. In the present study, phytochemical analysis of ethanol extracts of B. flabellifer sprout peel showed the presence of flavonoids. About 500 µg of our extract showed 59.8% of breast cancer cell inhibition. The same results were supported by Sundarraj et al.[31]

Friedman et al. reported the antibacterial activities of phenolic benzaldehydes and benzoic acids against Campylobacter jejuni, E. coli, Listeria monocytogenes, and Salmonella enterica.[32] Similarly, in the present study, ethanol extract of palmyra palm sprout peel, GC-MS analysis showed a high number of benzoic acids that may contribute to the antibacterial activities against UTI bacteria. Rajeswari et al. reported, unsaturated alcoholic compound, 9,17-octadecadienal, (Z)-, to have antimicrobial property.[33] Methyl ester, and 9,12-octadecadienoic acid (Z, Z)-, possess anti-inflammatory and cancer preventive characteristics. In this study, the higher antioxidant and antimicrobial activities of the ethanol extract of palmyra palm sprout peel could be due to the presence of a greater number of bioactive compounds such as n-hexadecenoic acid, phytol, and squalane. Hexadecanoic acid is known to exhibit strong antimicrobial and anti-inflammatory activity.[34] Likewise, in the present study, stigmasterol was shown to possess better antimicrobial and antioxidative properties. This could be one of the reasons for the higher antimicrobial activity exhibited by ethanol extract from the palmyra palm sprout peel.

γ-sitosterol reduces the hyperglycemia in STZ-induced diabetic rats due to increased insulin secretion and inhibition of glucogenesis. According to studies, it can be used in the treatment of diabetes mellitus.[35] In our study, ethanol extract of ethanol from the palmyra palm sprout peel showed antidiabetic activity via inhibition of alpha amylase. Docking studies of the ligand ã sitosterol with four different target proteins showed that this is a good molecule which docks well with various targets related to Diabetes mellitus, thus γ-sitosterol can be considered for developing into a protein antidiabetic drug.[36] Sundarraj et al. reported that γ-sitosterol may affect the amount and activity of components of the extrinsic apoptic pathway in human lung and breast adenocarcinoma cells.[31]

CONCLUSION

This study concluded that ethanol extract of the palmyra palm sprout peel showed excellent antioxidant, anti-inflammatory, antidiabetic, and antiproliferative activities. Before boiling the Panam kilangu, the sprout skin is usually removed from it and thrown as waste. The abundant and cheap source of natural waste product is used as an alternative source to obtain medicinal compounds and also to evaluate pharmacological active compounds of its constituents in a greater deal for ensuring safety use in our study. The ethanolic extract of the palmyra palm sprout peel has shown exploitable medicinal properties which may revolutionize the world of natural medicine 1 day. This study also shows the significance of plant-based products, which may be conventionally considered as waste, may also possess invaluable benefits.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.