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Clinical Phytoscience

International Journal of Phytomedicine and Phytotherapy

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Screening of phytochemical and pharmacological activities of Syzygium caryophyllatum (L.) Alston

Clinical Phytoscience volume 4, Article number: 3 (2018) Cite this article

Abstract

Background

The systematic screening of plant species with the purpose of discovering new bioactive compounds is prerequisite for any bioprospecting study. Therefore the present study was carried out to assess the phytochemical content and the evaluation of in vitro antioxidant, antibacterial, larvicidal and antidiabetic activities of the methanol extracts of S.caryophyllatum bark, leaves, fruit pulp and seeds.

Methods

The quantitative estimation of total phenol, flavonoid and tannin content of the extracts of S. caryophyllatum bark, leaf, fruit pulp and seeds were analyzed by using standard methods. The present study also conducted to screen antioxidant (DPPH, ABTS assays), antimicrobial, antidiabetic (in vitro α-amylase and α-glucosidase inhibitory assays) and larvicidal (against fourth instars larvae of Aedes aegypti and Culex quinquefasciatus) activities of methanol extract of S. caryophyllatum.

Results

The results of quantitative phytochemical analysis revealed the presence of maximum amount of phytoconstituents such as phenol, flavonoids and tannins in the leaf, bark and seed extract when compared to fruit pulp extracts. Free radical scavenging activity indicated that methanol bark, leaves, fruit and seeds extracts has significant free radical scavenging ability on DPPH with percentage inhibition of 88.15%, 81.31%, 75.24% and 83.36% respectively. The in vitro α-amylase and α-glucosidase inhibitory studies of the methanol crude extracts of four different plant parts of S. caryophyllatum (Bark, leaf fruit pulp and seed) showed good inhibitory activities in concentration dependent manner. The maximum percentage inhibitory activity of 78.03% was showed at concentration of 500 μg/ml seed extracts followed by bark (78.03%), leaf (69.4%) and fruit pulp (56.9%) at the same concentration. While the percentage inhibitory activity of four extracts also showed potent inhibition of α-glucosidase; maximum inhibition exhibited at the concentration of 100 μg/ml by bark extract (80.9%) compared with other extracts, leaf (78.2%), seed (77.59%) and fruit pulp (63.35%). The leaf essential oil and four extracts showed significant mortality against fourth instars larvae of Ae. aegypti and Cx. quinquefasciatus respectively.

Conclusion

Thus the present study suggests that S.caryophyllatum plant parts can be used as natural antioxidant source to prevent diseases associated with free radicals. Also, this plant can be a good source for further purification studies for isolation and characterization of compounds related to these antioxidants, antidiabetic and antibacterial activities.

Background

Nature has been a resource of medicinal treatments for thousands of years, and plant derived products being continued to play a crucial role in primary health care of approximately 80% of the world’s population [1]. Plant based drugs can be derived from any part of the plant like leaves, bark, flowers, fruits, root etc. i.e., any part of the plant may contain active components. The systematic screening of plant species with the purpose of discovering new bioactive compounds is prerequisite for any bioprospecting study. The fruits from wild edible plants possess significant amount of vitamins (β-carotene, vitamins C and E), flavonoids and other polyphenolic compounds having high free-radical scavenging activity [2]. An epidemiological study provides convincing evidence of the beneficial role of fruits and vegetables in the diet for the maintenance of health and prevention of diseases [3]. Polyphenols represent a diverse class of compounds including flavonoids (i.e. flavones, flavonols, flavanones, flavononols, flavan-3-ols and chalcones), tannins and phenolic acids [4]. These polyphenols are products of secondary metabolism in plants, and their important role of chelating transitional metals and scavenging free radicals activity are mainly due to their redox properties and chemical structures [5]. Antioxidants are vital substances which possess the ability to protect the body from tissue damage caused by free radical induced oxidative stress [6]. In addition to common antioxidants like vitamin C, vitamin E and carotenoids, phenolic constituents such as flavonoids, tannins and anthocyanins present in food of plant origin are also potential antioxidants [7,8,9].

Plant phenolics present in the fruit and vegetables have received considerable attention because of their potent antioxidant property. Plants possess more phenolic content show better antioxidant activity this is due to the direct correlation between total phenolic content and antioxidant activity [10, 11]. Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanism of action [12, 13]. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials [14].

Mosquitoes are the most important single group of insects in terms of public health importance, which are responsible for spreading a number of severe diseases, such as malaria, filariasis, dengue, Japanese encephalitis, etc. Culex quinquefasciatus is a major contributor for transmitting vector borne diseases like fevers and filariasis to human beings and animals. More than 40 million peoples affected globally from Elephantiasis and are badly debilitated and disfigured from this disease [15]. Aedes mosquitoes on the other hand are vectors of important viral diseases of man such as the zika virus, dengue and yellow fevers. Ae. aegypti is reported to be the principal vector of dengue fever in tropical countries, a disease considered a serious public health problem world over, especially in areas where the environmental conditions for the development of Aedes mosquitoes abound. Totally, 2.35 million people suffering from dengue was reported in America alone (as per 2013 data) of which 7687 cases were rigorous [16]. According to Directorate of National Vector Borne Disease Control Programme (NVBDCP), till July 30, 2016, the total dengue cases in India were 16,870 while for the same period in 2017 they numbered 28,702. Recently, the infections have been raised due to an increased urbanization, trade, and travel. No effective drug or vaccine is available so far. Frequent uses of these synthetic insecticides create an increase resistance among the mosquitoes and to cause adverse impacts on non-target organisms or humans. With this scenario, biological control especially plant based insecticide has proven as better in controlling insect vectors.

Plant extracts have long been used for the treatment of diabetes in various traditional systems of medicine and are now accepted as an alternative for diabetic treatment. Type II diabetes mellitus is a chronic endocrine metabolic disorder that affects the carbohydrates, proteins and fat metabolism. Postprandial increase of blood glucose is the major problem in diabetic patients. Inhibitors of α-amylase and α-glucosidase enzymes delay the breaking down of carbohydrates in the small intestine and decrease the postprandial blood glucose levels. Inhibitors of α-amylase and α-glucosidase isolated from medicinal plants to serve as an alternative drug with increased potency and lesser adverse effects than existing synthetic drugs.

Syzygium caryophyllatum (L.) Alston., an endangered evergreen tree belongs to the family Myrtaceae, commonly known as Wild black plum. This tree is native to Sri Lanka and India; in India the distribution is restricted to the forests of Western Ghats regions. S. caryophyllatum fruits are edible, sweet and astringent in taste. The seeds and bark were dried and its decoction was used in the ailment of diabetes mellitus [17]. The leaf and bark extracts of this plant are well known for its antibacterial and antioxidant efficacy [18]. Root extract using different solvent system exhibited promising amount of phenol, flavonoids, tannins and vitamin C [19]. The bark extract of this plant was used in veterinary medicine for the treatment of tympanitis in cattles [20]. Tribal people consider this plant as a boon of nature and its fruits and seeds were consumed by Paniya tribal community of Waynad district, Kerala, India [21]. As the plant is used for medicinal purposes, the investigation of chemical constituents in the plant parts and its biological activities may provide valuable information. There is a detailed bioprospecting studies by using different organic solvent system with different plant parts have not been studied previously on S. caryophyllatum. Therefore the present study was carried out (i) to analyse the leaves essential oil and its antibacterial and larvicidal activities (ii) to assess the total phenols, flavonoids and tannins content; (iii) to evaluate the in vitro antioxidant, antibacterial, larvicidal and antidiabetic activities of the methanol extracts of S.caryophyllatum bark, leaves, fruit pulp and seeds.

Methods

Plant material and sample preparations

S. caryophyllatum plant samples (Leaves, bark and fruits) were collected from Kannur district of Kerala, India during 2012 and voucher specimen (PLS2011007) was prepared, identified and deposited at the Department of Plant Science herbarium, Madurai Kamaraj University. The bark, leaves, fruit pulp and seeds were separately dried at below 40 °C. Powdered sample of S.caryophyllatum bark, leaves, fruit pulp and seeds was sequentially extracted with hexane, ethyl acetate, methanol and water in their increasing polarity by sonicator. The extracts were filtered through Whatman No.1 filter paper and the solvents were evaporated under reduced pressure. Dried extracts obtained were stored in 4 °C until further analysis.

Estimation of total phenol content

The amount of total phenol in plant extracts was determined with the Folin–Ciocalteu reagent using the method given by Lister and Wilson [22]. 100 μl of appropriate dilutions of extracts were dissolved in 500 μl (1/10 dilution) of the Folin–Ciocalteu reagent and 1000 μl of distilled water. The solutions were mixed and incubated at room temperature for 1 min. After 1 min, 1500 μl of 20% sodium carbonate (Na2CO3) solution was added to the mixture and then incubated for 2 h in the dark at room temperature. A standard curve was prepared by using gallic acid as a standard. Different concentrations of gallic acid were prepared in 80% of methanol. The absorbances of all samples were measured at 760 nm using a UV-Vis spectrophotometer (Model. U.2800, Hitachi) and the total phenol contents were expressed as mg/g gallic acid equivalent (GAE) using the equation obtained from the calibration curve using the following eqs.

$$ Y=b\times +c $$
(1)

Where, Y-absorbance of extract; c-intercept; b-slope of the calibration curve; x- concentration of extract.

$$ X=Y-c/m $$
(2)
$$ C= cV/m $$
(3)

Where, C-Total phenolic content; c-concentration of gallic acid obtained from calibration curve in mg/ml; V-volume of extract in ml; m- mass of extract in gram.

Estimation of total flavonoid content

Estimation of total flavonoid content in the dried extracts was determined spectrophotometrically following the method given by Quettier-Deleu et al. [23]. This method is based on the formation of a complex flavonoid–aluminium, having the absorbtivity maximum at 430 nm. 1 ml of diluted sample was separately mixed with 1 ml of aluminum chloride solution (2% w/v). After incubation at room temperature for 15 min, the absorbance of the reaction mixture was measured at 430 nm. Rutin was used to make the calibration curve. The flavonoid content was expressed in mg per g of rutin equivalent (RE using the equation obtained from the calibration curve using the following eqs.

$$ Y= bx+c $$
(1)

Where, Y-absorbance of extract; c-intercept; b-slope of the calibration curve; x- concentration of extract.

$$ X=Y-c/m $$
(2)
$$ C= cV/m $$
(3)

Where, C-Total flavonoid content; c-concentration of rutin obtained from calibration curve in mg/ml; V-volume of extract in ml; m- mass of extract in gram.

Estimation of tannin content

The amount of tannin content was determined by modified Prussian blue method [24]. This method is based on the mechanism that the phenols reduce potassium ferricyanide to produce ferrous ions; these ferrous ions in turn react with ferric chloride in the presence of dilute HCl to form a Prussian blue coloured complex, which can be measured spectrophotometrically at 700 nm wavelength. 100 μl of appropriate dilutions of extracts were dissolved in distilled water to make up the volume of 7 ml. The sample solution was mixed with 1 ml potassium ferricyanide followed by the addition of 1 ml ferric chloride. The absorbance of samples was measured at 700 nm. Tannic acid was used to make a calibration curve. The amount of tannin in plant extracts was calculated by using the equation obtained from the calibration curve using the following eqs.

$$ Y=\mathrm{bx}+\mathrm{c} $$
(1)

Where, Y-absorbance of extract; c-intercept; b-slope of the calibration curve; x- concentration of extract.

$$ X=Y-c/m $$
(2)
$$ C= cV/m $$
(3)

Where, C-Total Tannin content; c-concentration of Tannic acid obtained from calibration curve in mg/ml; V-volume of extract in ml; m- mass of extract in gram.

In vitro free radical scavenging assays

Free-radical scavenging ability (DPPH-assay)

The scavenging ability of leaf essential oil, bark, leaves, fruit pulp and seed extracts on DPPH free- radicals was estimated according to the method of Shimada et al. (1992) [25]. This method depends on the reduction of purple 1,1-diphenyl-2-picrylhydrazyl (DPPH) to a yellow coloured diphenyl picrylhydrazine and the remaining DPPH which showed maximum absorption at 517 nm was measured. About 2 ml of various concentrations of test samples and ascorbic acid and gallic acid standards (2.5–20 μg/ml) were mixed with 0.5 mL of 1 mM DPPH in methanol. An equal amount of methanol and DPPH served as control. The mixture was shaken vigorously and then incubated for 30 min at room temperature in dark. The absorbance of the samples/standards was measured at 517 nm against the blank. The decrease of the absorbance of the DPPH solution indicates an increase in DPPH radical scavenging activity. The experiment was performed in triplicates. The DPPH radical scavenging activity was calculated according to the following equation:

$$ \%\mathrm{DPPH}\ \mathrm{radical}\ \mathrm{scavenging}\ \mathrm{activity}=\left(\mathrm{Ao}\hbox{-} \mathrm{A}1\right)/\mathrm{Ao}\times 100\%, $$

where Ao is the absorbance of the control reaction and A1 is the absorbance in the presence of the sample of the tested extracts. Percentage radical activity was plotted against the corresponding antioxidant substance concentration to obtain the IC50 value, which is defined as the amount of antioxidant substance required to scavenge the 50% of free radicals present in the assay system. IC50 values are inversely proportional to the antioxidant potential.

ABTS radical cation scavenging assay

ABTS radical cation scavenging assay was carried out by the method of Re et al. [26]. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS2•) radical cation was generated by adding 14 mM ABTS to 4.9 mM ammonium persulphate and the mixture was allowed to stand for 12–16 h in the dark at room temperature. This solution was diluted to obtain an absorbance of 0.7 ± 0.05 with ethanol at 734 nm (Shimadzu UV-Vis spectrophotometer model 2450) and the same was used for further assay. To 900ul of ABTS radical solution, added 100ul of the different concentrations of extracts (100-1000μg/ml) and ascorbic acid and gallic acid standards (2.0–10 μg/ml). The solution was shaken well and the absorbance was measured after 7 min. The capacity to scavenge the ABTS radical cation was calculated using the formula same as DPPH method.

In vitro antidiabetic assays

α-amylase inhibition assay

The α-amylase inhibitory activity of the methanol extracts of bark, leaves, fruit pulp and seed of the S.caryophyllatum was determined according to the method described by Ranilla et al. [27]. A total of 250 μL of sample/standard and 125 μL of 0.02 M sodium phosphate buffer (pH 6.9 with 6 mM NaCl) containing α - amylase solution (0.5 mg/mL) was incubated at 25 °C for 10 min. After pre-incubation, 250 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 6 mM NaCl) was added to each tube at timed intervals. The reaction mixtures were then incubated at 25 °C for 10 min. The reaction was stopped with 0.5 mL of dinitrosalicylic acid colour reagent. The test tube was then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 5 mL of distilled water and absorbance was measured at 540 nm. Acarbose was used as the positive control. The α -amylase inhibitory activity was calculated as follows;

$$ \% Inhibition\ of\alpha - amylase=\left( Ao\hbox{--} A1\right)/ Ao\times 100\%. $$

Where, Ao is the absorbance of the control reaction and A1 is the absorbance in the presence of the sample of the tested extracts.

α-glucosidase inhibition assay

The α-glucosidase inhibitory activity was assessed by Dong et al. [28] with slight modifications. Briefly, a volume of 60 μL of sample/acarbose standard solution and 50 μL of 0.1 M phosphate buffer (pH 6.8) containing α-glucosidase solution (0.2 U/mL) was incubated in 96 well plates at 37 °C for 20 min. After pre-incubation, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside (PNPG) solution in 0.1 M phosphate buffer (pH 6.8) was added to each well and incubated at 37 °C for another 20 min. Then the reaction was stopped by adding 160 μL of 0.2 M Na2CO3 into each well, and absorbance was recorded at 405 nm by micro-plate reader and compared to a control which had 60 μL of buffer solution in place of the extract. The α-glucosidase inhibitory activity was expressed as percentage inhibition and was calculated same as α-amylase.

Antibacterial sensitivity tests

Antibacterial activity of bark, leaves, fruit pulp and seed extracts were investigated by the disc diffusion method [29]. Antibacterial activity was carried out against six bacteria viz., Bacillus cereus, B. licheniformis, Escherichia coli, Staphylococcus aureus, S. hominis and Aerococcus viridians. Mueller-Hinton agar medium (MHA) was used for antibacterial susceptibility tests. Petri plates were prepared by pouring 20 mL of MHA medium and allowed to solidify. The bacterial suspensions were then streaked over the media surface using a sterile cotton swab to ensure confluent growth of the organism. 20 μL of 2.5 mg /mLof bark, leaves, fruit pulp and seed extracts were impregnated on sterile discs of 6 mm size. The discs were then aseptically placed on the surface of agar plates at well-spaced intervals. The plates were incubated at 37 °C for 24 h and the observed zones of inhibition were measured. DMSO serve as a control and antibiotic discs (6.0 mm in diameter) of 10/10 μg/mL Ampicillin/Sulbactum, 120 μg/mL Gentamycin were also used as positive controls. Antibacterial activity was evaluated by measuring the diameter of the zone of inhibition against tested bacteria.

The minimum inhibitory concentration (MIC) values were determined for the bacterial strains which were sensitive to the bark, leaves, fruit pulp and seed extracts in disc diffusion assay. The inoculums of the bacterial strains were prepared from 12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The bark, leaves, fruit pulp and seed extracts of S. caryophyllatum dissolved in 5% dimethyl sulfoxide (DMSO) were first diluted to the highest concentration (1000 μg/mL) to be tested, and then serial twofold dilutions were made in order to obtain a concentration range from 62.5–1000 μg/mL in 10 mL sterile test tubes containing nutrient broth. MIC values of S. caryophyllatum bark, leaves, fruit pulp and seed extracts against bacterial strains were determined based on a microwell dilution method. The tubes were observed after incubation of 24 h at 37 °C. After incubation, the MIC of each sample was determined by visual inspection of the tubes. The lowest concentration of the active ingredient that inhibited growth, as detected by a lack of visual turbidity was taken to be the MIC.

Larvicidal activity

The larvicidal activity of the leaf essential oil and methanol extracts of bark, leaf, fruit pulp and seed of S. caryophyllatum were tested according to the WHO [30] procedure with slight modifications. Larvicidal activity was tested against two mosquito vectors namely Aedes aegypti (Ae. aegypti) and Culex quinquefasciatus (Cx. quinquefasciatus). The larvae were obtained from Centre for Research in Medical Entomology, Madurai. The essential oil was dissolved in methanol to make the stock solution of 10,000 ppm (10 μL/1 mL). From the stock solution, concentration of 10, 50, 100, 200 and 400 ppm were prepared. The 1% stock solution of crude extracts of bark, leaf, fruit pulp and seed were prepared with methanol. From the stock solution, 500–62.5 mg/L, dilutions were prepared with methanol. Twenty early fourth instars larvae were introduced in insects breeding box containing 100 mL of water with each concentration. A control was prepared by the addition of methanol. Larvae were fed a diet of dry yeast and dog biscuits. The numbers of dead larvae were counted after 24 h of exposure and the percentage mortality was reported from the average of three replicates. The average larval mortality data were subjected to probit analysis for calculating LD50 and LD90 values were calculated by using the software BioStat, 2009.

$$ \mathrm{Percent}\ \mathrm{mortality}=\%\mathrm{Mortality}\ \mathrm{in}\ \mathrm{treated}\hbox{--} \%\mathrm{Mortality}\ \mathrm{in}\ \mathrm{control}/100\hbox{--} \%\mathrm{Mortality}\ \mathrm{in}\ \mathrm{Control} $$

Statistical analysis

All data were reported as mean ± standard deviation/standard errors of means of three replicates. The IC50 values were calculated using the ED50plus v 1.0 programme. Statistical analyses were performed using Microsoft Excel.

Results

The quantitative estimation of total phenol, flavonoid and tannin contents of different solvent extracts of S. caryophyllatum bark, leaf, fruit pulp and seed were analysed. The concentration of total phenol content expressed in terms of gallic acid equivalent (GAE) (the standard curve equation: y = 0.188× + 0.030, R2 = 0.997). The concentrations of total phenol content in bark, leaf, fruit pulp and seed extracts of S. caryophyllatum ranged from 0.74 to 36.1 mg GAE/g dry weight. The highest amount of total phenol was found to be in methanol extracts of leaf (36.1 mg GAE /g), bark (31.6 mg GAE /g) and seed (29.1 mg GAE /g). While ethyl acetate extracts of leaf, bark and seed were also exhibited significant amount of phenol content with 25.7, 17.8 and 22.28 mg GAE/g respectively. The lowest concentration of total phenol content was obtained in fruit pulp extracts (Table 1).

Table 1 The quantitative estimation of total phenol content of the different solvent extracts of S. caryophyllatum leaf, bark fruit pulp and seed
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The concentration of total flavonoid content was expressed in terms of (RE) rutin equivalent (the standard curve equation: y = 0.175×-0.029, R2 = 0.994). The concentration of flavonoid content in bark, leaf, fruit pulp and seed extracts of S. caryophyllatum ranged from 0.51 to 5.63 mg RE/g dry weight. The highest concentration of flavonoid level was obtained in methanol extract from the leaf (5.63 mg RE/g) followed by bark methanol (3.6 mg RE/g) and leaf water extract (3.75 mg RE/g) respectively. Fruit pulp and seed extracts of all solvent fractions showed significant amount of flavonoid content. The lowest concentration of flavonoid was found in hexane and ethyl acetate extracts of leaves and bark (Table 2).

Table 2 The quantitative estimation of total flavonoid content of the different solvent extracts of S. caryophyllatum leaf, bark fruit pulp and seed
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Total tannin content in bark, leaf, fruit pulp and seed extracts were expressed in terms of (TAE) tannic acid equivalent (the standard curve equation: y = 0.229× + 0.041 R2 = 0.998). The maximum concentration of total tannin content was found to be 36.6 mg TAE/g in methanol extract of leaf followed by methanol bark extract (31.3 mg TAE/g), water leaf extract (26.3 mg TAE/g), ethyl acetate leaf extract (25.3 mg TAE/g) and 22.9 mg TAE/g in methanol extract of seed. Among the four plant parts tested, lowest tannin content was obtained in fruit pulp extracts (Table 3). The results of quantitative phytochemical analysis revealed that the presence of greater amounts of phytoconstituents such as phenol, flavonoids and tannins in the methanol extracts leaf, bark, fruit pulp and seed of S. caryophyllatum when compared to other solvent extracts.

Table 3 The quantitative estimation of total tannin content of the different solvent extracts of S. caryophyllatum leaf, bark fruit pulp and seed
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Antibacterial sensitivity test

The antibacterial activity of crude methanol extracts of bark, leaves, fruit pulp and seed was determined by disc diffusion and minimum inhibitory concentration methods against six bacterial species viz. Bacillus cereus, B. licheniformis, Staphyllococcus aureus, S. hominis, Aerococcus viridians and Escherichia coli. Methanol bark extract exhibited the pronounced antibacterial activity against S. hominis, B. licheniformis and A. viridian with zone diameter of 12.3 mm, 12 mm and 11.3 mm respectively. Leaf extract showed potent antibacterial activity against E. coli (14.3 mm), B. cereus (14 mm), S. aureus (12.3 mm) but it did not exhibited any zone of inhibition against S. hominis. Methanol seed extract exhibited the pronounced antibacterial activity against A. viridian, B. cereus and S. aureus with zone diameter of 12 mm, 13 mm and 10 mm respectively (Table 4). However, fruit pulp extract exhibited moderate effect of antibacterial activity against some of the tested bacterial strains. The maximum zone of inhibition showed against S. aureus with zone diameter of 9 mm. The blind control (DMSO) did not inhibit any of the bacteria tested. The MIC values confirmed the significant activity against the tested microorganisms, as shown in Table 5.

Table 4 Antibacterial activities of leaf, bark, fruit pulp and seed methanol extracts of S. caryophyllatum
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Table 5 Minimum inhibitory concentration (MIC) of leaf, bark, fruit pulp and seed methanol extracts against pathogenic bacteria
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Larvicidal activity

Results obtained in the present investigation on the leaf essential oil and methanol crude extracts of bark, leaf, fruit pulp and seed of S. caryophyllatum exhibited potent larvicidal activity in terms of LD50 and LD90 against the two selected mosquito vectors Aedes aegypti and Culex quinquefasciatus are presented in Table 7. The leaf essential oil of S. caryophyllatum showed 100% mortality against fourth instar larvae of Ae. aegypti and Cx. Quinquefasciatus at concentration of 400 ppm. At 200 ppm, over 95% mortality of fourth instar larvae of Ae. aegypti and 81% mortality of Cx. Quinquefasciatus were observed. The LD50 values of the leaf essential oil recorded after 24 h of exposure were 54.8 ppm and 95.26 ppm against Ae. aegypti and Cx. quinquefasciatus respectively. The larvicidal activity increased with an increase in the leaf essential oil concentration. Similar trends were observed in the methanol crude extracts of bark, leaf, fruit pulp and seed; and the results of mean percentage mortality against two selected mosquito vectors in different concentrations were also shown in Table 6. The methanol extracts of all plant parts exhibited lethal effects on Ae. aegypti and Cx. quinquefasciatus after 24 h of exposure at different concentrations. The highest mortality rate was found to be in methanol extract of leaf compared with other plant parts. The methanol extracts of leaf, bark and seed extracts showed over 80% mortality at 500 mgL. Leaf methanol extract of S. caryophyllatum was effective against the fourth instar larvae of Ae. aegypti and Cx. quinquefasciatus with LD50 values of 80.4 (LD90 = 346.5 mg/L) and 128.1 mg/L (LD90 = 363.9 mg/L) respectively. The bark and seed methanol extracts had significant larvicidal effect with LD50 and LD90 values found to be 303.6 (LD90 = 541.3 mg/L) and 286.0 (LD90 = 554.8 mg/L) against Ae. aegypti and 286.4 (LD90 = 509.5 mg/L) and 300.2 (LD90 = 547.2 mg/L) against Cx. quinquefasciatus. Methanol fruit pulp showed the least larvicidal activity compared with other three plant parts studied. At 500 mg/l concentration, it caused 71.6% and 48.3% larval mortality against Ae.aegypti and Cx. quinquefasciatus with LD50 and LD90 values of 387.0 (LD90 = 651.6 mg/L) and 519 mg/l (LD90 = 876.1 mg/L) respectively. The LD50 and its lower, upper confident levels, along with the regression analyses are given in Table 7. The results obtained in this study clearly proved that the S. caryophyllatum leaf essential oil and effective constituents of methanol extract of different plant parts could be considered as a potent source for the production of eco-friendly larvicidal agent against the disease causing common mosquito vectors.

Table 6 Larvicidal activity indifferent concentrations of S. caryophyllatum plant extracts and essential oil against Ae. aegypti and Cx. quinquefasciatus (mg/l)
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Table 7 LD50 and other statistical analysis of S. caryophyllatum plant extracts and essential oil against fourth instar larvae of Ae. aegypti and Cx. quinquefasciatus
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Free radical scavenging assays

DPPH is a stable free radical, which has been widely accepted as a tool for estimating free radical-scavenging activities of antioxidants. The percentage inhibition of DPPH and ABTS in the methanol extracts of bark, leaves, fruit pulp and seeds at different concentrations are shown in Figs. 1 and 2. The method is based on the reduction of methanolic-DPPH solution because of the presence of antioxidant substances having hydrogen donating groups (RH) such as phenolics and flavonoids compounds due to the formation of non radical DPPH-H form. Free radical scavenging activity indicated that methanol bark, leaves, fruit and seeds extracts have significant free radical scavenging ability on DPPH with percentage inhibition of 88.15%, 81.31%, 75.24% and 83.36% respectively. The four extracts also revealed scavenging effects on ABTS with percentage inhibition of 88.15%, 81.31%, 75.24% and 83.36% respectively. The IC50 values of DPPH and ABTS radical activities of the methanol extracts of bark, leaves, fruit pulp, seeds and positive controls were shown in Table 8. A lower IC50 value indicates higher antioxidant activity. The results revealed that free radical scavenging activity of methanol fruit and leaves extracts has less radical scavenging ability on DPPH with IC50 value of 69.41 μg/mL, while the bark and seeds methanol extracts showed IC50 value of 48.37 μg/mL compared to the positive control ascorbic acid (IC50 = 9.55 μg/mL) and gallic acid (IC50 = 7.05 μg/mL). The ABTS radical activities of the methanol extracts of bark, leaves, fruit pulp and seed showed IC50 values of 10.26 μg/mL, 13.2 μg/mL, 69.4 μg/mL and 20.1 μg/mL respectively. The positive control ascorbic acid and gallic acid showed the IC50 values of 5.6 μg/mL and 4.9 μg/mL respectively.

Fig. 1
figure 1

DPPH free radical scavenging activity of S.caryophyllatum a Ascorbic acid; b Gallic acid; c Bark methanol; d Leaf methanol; e Fruit pulp methanol; f Seed methanol

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Fig. 2
figure 2

ABTS free radical scavenging activity of S.caryophyllatum a Ascorbic acid; b Gallic acid; c Bark methanol; d Leaf methanol; e Fruit pulp methanol; f Seed methanol

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Table 8 Free radical scavenging activities of S. caryophyllatum bark, leaf, fruit pulp, seed methanol extracts and standard antioxidants (Ascorbic acid and Gallic acid)
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In vitro α-amylase and α-glucosidase assays

The in vitro α-amylase and α-glucosidase inhibitory studies of the methanol crude extracts of four different plant parts of S. caryophyllatum (Bark, leaf fruit pulp and seed) showed good inhibitory activities in concentration dependent manner. Percentage inhibition was tested with different concentrations of four methanol extracts 100-500 μg/ml for α-amylase and 20-100 μg/ml for α-glucosidase. The bark extract indicated a potent inhibitory activity of α-amylase followed by seed, leaf and fruit extracts. The maximum percentage inhibitory activity of 78.03% was showed at concentration of 500 μg/ml seed extracts followed by bark (78.03%), leaf (69.4%) and fruit pulp (56.9%) at the same concentration. While the percentage inhibitory activity of four extracts also showed potent inhibition of α-glucosidase. The results showed that maximum inhibition exhibited at the concentration of 100 μg/ml by bark extract (80.9%) compared with other extracts, leaf (78.2%), seed (77.59%) and fruit pulp (63.35%). The percentage inhibition and IC50 values obtained in the analyses were shown in Fig. 3a, b and Table 8.

Fig. 3
figure 3

a In vitro alpha amylase activity of S. caryophyllatum. b In vitro alpha-glucosidase activity of S. caryophyllatum

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Discussion

Phytochemical screening provides fundamental information about the medicinal importance of the plant extracts. Phenolic compounds play an important role in plant defence mechanisms against invading bacteria and other types of environmental stress, such as wounding and excessive light or ultraviolet radiation [31, 32]. These compounds can act as antioxidants by chelating metal ions, preventing radical formation and improving the antioxidant endogenous system [33]. Antioxidants which predominantly originate from phytochemicals have been reported to play an important role in the treatment of diabetes [34]. Our results also in accordance with previous report that showed high phenol content in S. aqueum leaf ethanol extracts may be the reason for its high anti-glycation activity [35].

Syzygium species are known to be very rich in flavonoids, tannins and other polyphenol derivatives [36,37,38,39]. Flavonoids, tannins, anthocyanins and other phenolic constituents present in bark, leaves and fruits are potential antioxidants. Flavonoids are known to regenerate the damaged pancreatic β-cells in the alloxan induced diabetic rats [40]. Tannins are considered nutritionally undesirable because they precipitate proteins, inhibit digestive enzymes and affect the absorption of minerals and vitamins. Tannins were reported to exhibit antiviral, antibacterial and anti-tumour activities. However, some kinds of tannins can reduce the mutagenicity of a number of mutagens and also exhibited antioxidant, anticarcinogenic and antimicrobial activities [41].

Food rich in antioxidants plays a crucial role in the prevention of various types of cancers, cardiovascular and Alzheimer’s diseases [42]. Antioxidants which predominantly originate from phytochemicals like flavonoids and phenols have been reported to play an important role in the treatment of diabetes [34] (Kim et al., 2011). Total antioxidant capacity may better reflect the health beneficial quality of foods than individual measurements due to the possible cooperative action of antioxidants [43]. Seeds of S. cumini one of the close related species of S.caryophyllatum have been reported to be rich in flavonoids, which account for the scavenging of free radicals and a protective effect on antioxidant enzymes and they have also been found to have high total phenolic content with significant antioxidant activity [44, 45]. The results obtained in present study are in agreement with previous studies which showed that high total phenolic content increases the antioxidant activity [46]. According to previous reports the close related species S. cumini, S. aromaticum fruits and leaves extract have been reported to have high antioxidant activity [47, 48].

Different parts of S. cumini such as seeds, bark, fruit and leaves have been used in traditional medicine as a remedy for diabetes mellitus in many countries [49, 50]. The use of alpha-amylase and alpha-glucosidase enzyme inhibitors could be a prospective therapeutic agent for the effective management of postprandial hyperglycemia in diabetes mellitus patients [51]. It is obvious that flavonoids and polyphenols have been shown to inhibit pancreatic alpha-amylase and intestinal alpha-glucosidase enzymes and also reported that there is a positive correlation between the inhibition of alpha-amylse and alpha- glucosidase and flavonoids and polyphenol content [52,53,54]. Our investigation of in vitro studies of bark, leaf, fruit pulp and seed methanol extracts of S. caryophyllatum also showed remarkable inhibition of alpha-amylase and alpha-glucosidase suggesting the presence of potential enzyme inhibiting compounds in the plant extracts.

The use of plant extract for mosquito control save the environment from synthetic chemicals. Plant products are emerging as a potential source of mosquito control and among them essential oil have special interest due to their insecticidal properties [55, 56]. In general essential oils from plant sources have been considered important natural resources to act as insecticides [57, 58]. Essential oils derived from various plants not only exhibit inhibitory activity against fungi, bacteria and dermites but also show strong mosquito larvicidal and repellent activities [59]. The finding of the present study compare well with previous finding of Bagavan and Rahuman [60] who reported that the hexane crude extract of S. aromaticum flower bud exhibited potent larvicidal activity against fourth instar of Anopheles vagus and Cx. vishnui. A similar study conducted by Fayemiwo et al. [61] reported that the flower bud oil of S. aromaticum was effective against the Ae. aegypti (LC50 = 92.56 mg/l) and Cx. quinquefasciatus (LC50 = 124.4 mg/l) larvae. Our results are also comparable to earlier reports of Kaushik and Saini [62] who observed that the highest mortality was found in methanol leaf extract of S. cumini against the larvae of Ae. aegypti. The results of the present study suggested that the larvicidal potential of leaf essential oil and plant extracts of S. caryophyllatum and emphasize need to incorporate them in vector control and management programmes.

Conclusion

Based on the results of bioprospecting and biological screening of S. caryophyllatum proved to be a good medicinal plant used for curing various ailments in different traditional systems of medicine. The results of bioprospecting investigation the various polyphenolic compounds present in the plant extracts attribute the antioxidant, alpha amylase, alpha glucosidase, antibacterial and larvicidal activities. Thus the present study suggests that S.caryophyllatum bark, leaf, fruit pulp and seed can be used as natural antioxidant source to prevent diseases associated with free radicals. Also, this plant can be a good source for further purification studies for isolation and characterization of compounds related to these antioxidants, antidiabetic and antibacterial activities. These findings justify and confirm the traditional uses of this plant. Further research is necessary in order to know all the active principles and their pharmacological properties.

Abbreviations

ABTS:

-2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

DMSO:

Dimethyl sulfoxide

DPPH:

1,1-diphenyl-2-picrylhydrazyl

GAE:

Gallic acid equivalent

IC50:

50% Inhibitory concentration

LD50:

50% Lethal Dose

MHA:

Mueller-Hinton agar medium

mM:

Milli molar

Na2CO3:

Sodium Carbonate

NaCl:

Sodium Chloride

PNPG:

p-nitrophenyl-α-D-glucopyranoside

RE:

Rutin equivalent

TAE:

Tannic acid equivalent

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Acknowledgments

The authors would like to express their gratitude to Centre for Research in Medical Entomology (CRME), Madurai, India for providing mosquitos eggs for larvicidal analysis.

Funding

First author thanks University Grant Commission for the award of meritorious fellowship. Corresponding author thanks partial financial support through University Grant Commission-Centre of Advanced Study (UGC-CAS), Department of Biotechnology- Interdisciplinary Programme of Life Sciences for Advanced Research (DBT-IPLS) and Department of Science and Technology (DST)-Purse programmes.

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  1. Department of Plant Science, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, -21, India

    Stalin N & Sudhakar Swamy P

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Dr. Stalin: Completed his PhD in Department of Plant Science, School of Biological Sciences, Madurai Kamaraj University, Madurai. His area of research is species distribution modelling, bioprospecting and conversation of selected medicinal plant through phenological studies and in vitro propagation.

Dr. P. Sudhakar Swamy: Professor, Department of Plant Science, School of Biological Sciences, Madurai Kamaraj University, Madurai. His area of specialisation is Forest ecology, invasion ecology, bioprospecting of medicinal plants and their conservation.

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N, S., P, S.S. Screening of phytochemical and pharmacological activities of Syzygium caryophyllatum (L.) Alston. Clin Phytosci 4, 3 (2018). https://doi.org/10.1186/s40816-017-0059-2

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