UV/V is Spectrophotometric Characterization of the Leaf Polyphenolics Content in Elaeocarpus tectorius and its Therapeutic Potential against Selected Urinary Tract Infection Pathogens

M. Ashwini Lydia, Suman Thamburaj, Gayathri Jagadeesan, Gayathri Nataraj, Kasipandi Muniyandi, Saikumar Sathyanarayanan, and Parimelazhagan Thangaraj

17.1 INTRODUCTION

Antibiotics are the major drugs that have been used to act against numerous infective contagious diseases for decades. The wide use of these has caused a certain resistance, which is a common phenomenon of the microorganisms that are actually the causative agents. This urged the health care organizations and pharmaceutical industries all over the world to develop plant-based herbal products and formulations, as there is a drift in strategy. Meanwhile, there are substantial alternative sources of natural anti-microbials from plants that possess different modes of actions. Of those, many of them have been used in traditional medicinal systems for centuries and were found to have competitive effects as compared to a few commercial synthetic antibiotics (Abdallah 2011). In spite of the advancement and immense success in the development of medicine, science, and technology, still the spread and control of infectious diseases undergo failure. The World Health Organization stated that infectious diseases are the second leading issue for the cause of deaths in the world (WHO 2002). The emergence and rapid spread of new infections have now caused a switchover to the use of natural products with plants as the raw source having antibiotic actions, as they are known to be the largest biochemical and pharmaceutical stores on our planet and also carry active biochemical compounds with anti-oxidant and anti-microbial properties (Blois 1958; Cowan 1999). The biological activity of the plants can be predicted with respect to their use by people in traditional indigenous systems of medicine.

Urinary tract infections are the most common microbial infection occurring in the urinary tract. Frequently, women suffer from this infection during menstruation and pregnancy. So it occurs more predominantly in women than men. It is approximated that about 35% of healthy women suffer with UTIs at various stages in their life. About 5% of women each year experience a UTI with the problems of painful urination (dysuria) and frequency (Nabbugodi et al. 2015). The major causative agent for a UTI is the uropatho- genic bacteria E. coli (Abraham and Miao 2015). The causative agents for community-acquired urinary tract infections are mostly the uropathogenic Escherichia coli from the gut of about 80%-85%, then Pseudomonas aeruginosa with about 11%, Staphylococcus aureus with 5%-10%, followed by Candida albicans the yeast fungal pathogen as 9%, and they were the test pathogens used for the study (Nicolle 2008; Salvatore et al. 2011). They act as hosts for the frequent causes of urinary infections. The increasing multidrug resistance of UTI-causing microbial strains has made the treatment of UTIs difficult and has led to the greater use of costly broad- spectrum synthetic drugs (Tenney et al. 2018). This problem needs an improved effort, which has resulted in the search for valuable microbicide agents to be used against UTIs that are caused by pathogenic microorganisms resistant to current antibiotic drugs (Toner et al. 2016).

Though synthetic antibiotics tend to act against the pathogens, medicinal plants that are traditionally used by healers are valued.

Elaeocarpus is the largest genus belonging to the Elaeocarpaceae family, consisting of mostly tree species. There are about 350 species estimated to exist, and they are widely spread in Southeast Asian countries. In India, generally this species grows in the Himalayan region and temperate zones of the Western Ghats. Elaeocarpus species have many biological active molecules such as indolizili- dine alkaloids, triterpenes, tannin such as geraniin and 3, 4, 5-trimethoxy geraniin, grandisines, rudrakine fatty acids, ellagic acid derivatives, cytotoxic compounds flavonoids, and quercitin (Muthuswamya 2014). E. tectorius is a species that is one among the traditional plants used by the tribal people of the Nilgiris District, which is well known for its unique vegetation and diversity. The indigenous traditional knowledge of the utilization of E. tectorius is used in the treatment of leprosy, pneumonia, rheumatism, ulcers, piles, and dropsy. In view of the above information, E. tectorius was taken as the experimental plant to seek out the variations of the total polyphenolic contents in the leaf extracts and their therapeutic potential. This is the first report of antimicrobial activity of this particular plant against selected UTI pathogens.

  • 17.2 MATERIALS AND METHODS
  • 17.2.1 Collection of Plant Leaves

The E. tectorius leaves were collected from Coonoor, The Nilgiris District, Tamil Nadu, India, and were then identified with their morphological characteristics and description by using the voucher specimen in the Botanical Survey of India Southern Regional Centre, Coimbatore, Tamil Nadu. The plant specimen was given the reference no. BSI/ SRC/5/23/2018/Tech./2853. The leaves were first washed with distilled water to remove the adhering external dusts. Then they were subjected to shade drying under normal environmental conditions and were further powdered finely using a blender.

17.2.2 Extraction and Processing

Extraction of the leaf sample in different solvents is important for carrying out further analysis and comparative studies. Soxhlet apparatus was used for extraction using solvents such as petroleum ether, dichloromethane, ethyl acetate, methanol, and water. One hundred milligrams of the leaf samples were extracted using 300 mL of each solvent. Each extract was thereafter concentrated using a rotary vacuum evaporator (Equitron Evll-ABS.051), and then air dried. The dried extracts were stored in small glass vials at 20°C for further studies. The amount of crude extracts recovered after successive extraction was weighed, and the yield percentage was calculated based on the following formula,

17.2.3 Preliminary Phytochemical Analysis

Plants are the biological factories containing various biochemical phytocompounds. The screening of those compounds is the important part of the featured study. The leaf powder of E. tectorius was screened for the presence of phytochemical constituents, such as alkaloids, phenolics compounds, tannins, flavonoids, terpenoids, saponins, glycosides, and steroids using standard methods (Thangaraj 2016).

17.2.4 Determination of Total Phenolic,

Flavonoid, and Tannin Contents

Anti-oxidant compounds such as phenolics flavonoids and tannins were estimated quantitatively. The total phenolic content of the E. tectorius leaf extracts was determined according to the method described by Siddhuraju and Becker (2003). Initially, 0.5 mL of the Folin-Ciocalteu reagent was added to the plant sample, followed by 2.5 mL of 5% sodium carbonate, it was incubated in the dark at 27°C for 40 minutes, and the blue absorbance was measured at 725 nanometers. For the tannins, the extracts were treated with polyvinyl polypyrrol- idone. The tannin content of the plant extract was calculated by the following formula (Siddhuraju and Manian 2007),

The results of the total phenolics and tannins were expressed in gallic acid equivalents. The flavonoid content of E. tectorius leaf extracts was quantified according to the method given by Zhishen et al. (1999). To the plant extracts, 150 pL of 5% sodium nitrite and 10% aluminum chloride was added. This was incubated at 27°C for 6-10 minutes, and then 2 mL of 4% sodium hydroxide was added, and the absorbance of pink color was measured at 510 nanometers. The results were expressed as rutin equivalents, and all the estimations were made in triplicates.

  • 17.2.5 In Vitro Antioxidant Assays
  • 17.2.5.1 DPPH Scavenging Assay

The anti-oxidant activities of the E. tectorius leaf extracts were determined using the standard protocols. The ability of the plant extracts to scavenge the stable free radical 2,2-diphenyl-l-picrylhydrazyl (DPPH) was evaluated by the method of Blois (1958) with minor modifications (Thangaraj 2016). Plant extracts were diluted to various concentrations, and the volume was adjusted to 100 pL using methanol. About 5 mL of a 0.1 mM methanolic solution of DPPH was added to the aliquots of different extracts of the plant sample and standard rutin. The complex was then incubated for 20 minutes at room temperature, which resulted in the delocalization of electrons and also gave rise to a violet color formation; the absorbance was measured at 517 nm. The DPPH percentage inhibition was estimated using the following formula:

where A0 and Al were the absorbances of the blank and extracts or the absorbance taken place before (A0) and after the reaction (Al), respectively.

17.2.5.2 Ferric Reducing/Antioxidant Power Assay

The ferric reducing powerof the plant leaf extracts was esti mated based on the standard protocol given by Pulido et al. (2000), the ability to reduce the TPTZ (2,4,6-tripyridyl-s-triazine) (TPTZ)-Fe (III) complex to TPTZ-Fe (II) complex. The ferric reducing anti-oxidant power (FRAP) reagent (900 pL), which is to be freshly used in contact with the sample (10 pL of aliquots of plant extracts, 1 mg/mL of respective organic solvents), is prepared by three constituents 10 mM TPTZ in 40 mM HC1, 20 mM FeCl,.6H:0, and 0.3 M acetate buffer (pH 3.6) in the necessary quantity. Then the mixed solution is subjected to incubation at 37°C for 30 minutes. The ferric reduction is then measured at 595 nm. The results were expressed as mM Fe (II) equivalent/g extract.

17.2.5.3 Phosphomolybdenum Assay

The assay to determine the total anti-oxidant capacity was analyzed by the standard method of Prieto et al. (1999). The standard used was ascorbic acid. About 1 mg per mL of the sample concentration of each solvent extract and the standard ascorbic acid were added with 3 mL of the reagent solution that consisted of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The mixture was then incubated in a water bath at 95°C for about 90 minutes, the green phosphomolybdenum complex formation was noted after the incubation, and it was allowed to cool until it reached room temperature; the absorbance of the mixture was then monitored spectrophotometrically at 695 nm against the reagent, which was taken as the blank. The results were expressed as mg ascorbic acid equivalents (AAE)/g extract.

  • 17.2.6 Anti-microbial Activity
  • 17.2.6.1 Test Microbial Pathogens

Escherichia coli (MTCC-433), Staphylococcus aureus (MTCC-737), Pseudomonas aeruginosa (MTCC-741), and Candida albicans (MTCC-227) were the pure cultures maintained using a nutrient agar medium for bacteria and a Sabouraud dextrose agar medium for fungi and used for the anti-microbial testing. They were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh.

17.2.6.2 Disk-Diffusion Agar Method

The modified disk diffusion agar method (Olurinola 1996) was followed to verify the anti-microbial activity of E. tectorius leaf extracts against selected bacterial and fungal pathogens. The sterile swabs were used to swab the bacterial cultures from the broth onto the plates containing Mueller Hinton agar medium, similarly the fungal yeast pathogen ‘Candida’ were swabbed in the plate containing Sabouraud dextrose agar medium. Wells were made on the seeded plates with the help of a sterilized cork borer (6 mm Hi-Media). The collected different samples were further dissolved in 4% Dimethyl sulfoxide (DMSO). The diluted samples (100 pL of 10 mg/mL concentration) were dispended into the wells, and the plates were incubated aerobically at 37°C for 18-24 hours for bacterial pathogens and 28°C for 48 hours for fungal pathogens. In the same way, positive and negative control wells were made with Ampicillin-15 pg (bacteria), Miconazole-30 pg (Candida), and 4% DMSO, respectively. The entire microbial assay was carried out under strict aseptic conditions. The zones of inhibition (mm) of the samples were measured after incubation, and the activity index was also calculated. Triplicates were maintained for each pathogen, and the readings were taken in three different fixed directions, and the average values were recorded.

17.2.6.3 Determination of Minimum Inhibitory Concentration and Minimum Bactericidal/Fungicidal Concentration of E. tectorius Leaf Extracts

The determination of the minimum inhibitory concentrations (MICs) were carried out in a E. tectorius leaf for serial extract in the broth dilution method, for those that showed a > 8 mm diameter zone of inhibition as determined through the well diffusion method. The extract with 5 mg/mL concentration was gradually diluted in the 900 (iL sterile Mueller-Hinton bacterial broth and Sabouraud dextrose broth for Candida sp. Then, 100 pL of the culture (1 x 10s CFU/mL) was placed in all the broths, and one was kept as the control for each test microorganism. The culture tubes were then incubated at 37°C. The OD values were taken using a spectrophotometer (Shimadzu UV-spectrophotometer, UV-1800) at 570 nm. From the respective values obtained, the percentage of growth inhibition and MICs were calculated against each test pathogen. By sub-culturing, the test dilution used in the MIC and the minimum bactericidal/fungicidal concentration (MBC/MFC) was determined, thereafter by centrifugation (Eppendorf centrifuge 5430 R), the cultured pellets were suspended in 100 pL of the sterile broth. The total suspension was swabbed onto the desired plates containing the media and allowed to incubate for a further 24-48 hours at 37°C, and then onto a fresh solid medium and incubated further for 24 hours. The concentration of the plant extract that completely killed the organisms was considered as the MBC/MFC (CLSI 2008, 2009).

17.2.7 Ultraviolet/Visible Spectral Measurement

Ultraviolet (UV)-visible spectroscopy is performed for the qualitative analysis of phyto-compounds in plant extracts and for the identification of certain classes of polyphenolic compounds. This technique is very simple compared to other techniques and not time-consuming. The UV-visible (Vis) spectra of the diluted E. tectorius ethyl acetate and methanol leaf extracts (100 pL from 1 mg/mL concentration of extracts) were measured in the range of 200-1000 nm using a UV-Vis double beam spectrophotometer 1800 PC (Shimadzu, Japan).

17.2.8 Thin Layer Chromatography Separation

The Silica gel 60 F254 (Merck, Germany) was used for the thin layer chromatography (TLC) analysis of E. tectorius ethyl acetate and the methanol leaf extract. The silica plates (8 x 3 cm size) were activated at 120°C for 2 minutes and then used. The extracts to be separated were spotted (5 pL from 1 mg/mL concentration of extracts) with a capillary tube, 1.0 cm above from the end of the silica gel plate, which was in contact with the solvent hexane: ethyl acetate: methanol (60:30:10%). After spotting, the TLC plates were kept in a chamber containing the solvent. A chromatogram was developed by the movement of the solute (mobile phase) across a thin layer of silica (stationary phase). After the development of a chromatogram, the resolved spots were revealed by detection under UV light at a longer wavelength (365 nm) and a shorter wavelength (254 nm), which helped to detect the compound (colored compounds and fluorescence compounds).

17.2.9 Statistical Analyses

The results were expressed as mean ± standard deviation (SD). The data were statistically analyzed using the SPSS version 17.0 by means of one way Analysis of Variance (ANOVA), followed by Duncan’s test for total polyphenolics and anti-oxidant studies. Mean values were considered statistically significant when p < 0.05.

  • 17.3 RESULTS AND DISCUSSION
  • 17.3.1 Extract Yield Percentage

The leaf samples of E. tectorius were extracted using Soxhlet apparatus in a successive method. The yield of crude extract varies from each solvent extraction; it is based upon the nature and polarity of the solvents. Among the five solvent extractions of E. tectorius leaf powder, the maximum amount of yield was obtained in the methanolic extracts of the leaf (18.09%) (Table 17.1), whereas the dichloromethane extract of the leaf (1.2%) revealed the minimum yield. As the yield of higher polar solvents is considerably high as compared to the lower polar solvents, it may indicate that E. tectorius may have active components, such as polyphenolics, anti-oxidants, and anti-microbials.

Soxhlet extraction has been used for over a century and is a standard technique that is widely used for extracting valuable bioactive compounds from various natural sources, especially plants (De Castro and Priego-Capote 2010). The extraction of the bioactive components from the plant materials is a most important standard method and the first step used in different commercial sectors, such as pharmaceutical, food, and chemical industries (Azmir et al. 2013).

TABLE 17.1

Yield of the E. tectorius Leaf Extracts

Plant Materials Used

Solvents

Yield Extracts (%)

Leaf powder

Petroleum ether

1.70

Dichloromethane

1.20

Ethyl acetate

6.72

Methanol

18.09

Aqueous

3.22

17.3.2 Preliminary Phytochemical Screening

Preliminary phytochemical screening has revealed the presence of phytochemicals considered as active chemical constituents. Important phytochemicals such as alkaloids, phenolics, tannins, flavonoids, terpenoids, glycosides, and steroids were present in the E. tectorius leaf (Table 17.2). The result of the preliminary phytochemical analysis shows that the plant is rich in secondary metabolites. Medicinal plants having alkaloids are used in medicines for reducing headaches and fevers (Okwu and Okwu 2004). The terpenoids that are present are reported to have anti-inflammatory, anti-malarial, inhibition of cholesterol synthesis, anti-oxidant, and antimicrobial properties (Mahato and Sen 1997). Steroids have been reported for their wound healing properties, these are anti-inflammatory, analgesic, and anti-oxidant (Ayinde et al. 2007). The presence of glycosides in whole leaves might play a role in the cardioprotective potential of E. tectorius (Brian et al. 1985). Further, the presence of different phytoconstituents in the plant materials may be responsible for anti-microbial, anti-oxidant, and other biological properties. The phytochemical screening may be useful for the

TABLE 17.2

Phytochemical Screening of E. tectorius

Phylochemical Constituents

Leaf Powder

Alkaloids

+++

Phenolic compounds

+++

Tannins

++

Flavonoids

+

Saponins

-

Glycosides

++

Terpenoids

++

Steroids

+++

Note: (+): presence of chemical compound; (-): absence of chemical compound; (+) < (++) < (+++): based on the intensity of characteristic color.

identification of the bioactive principles and may lead to drug discoveries. Further, these tests facilitate their quantitative estimation and qualitative separation for pharmacologically active chemical compounds (Parivuguna 2008). The present study that was undertaken revealed the presence of a number of bioactive compounds which can be used as a lead compound for synthesizing drugs for various ailments.

17.3.3 Total Polyphenolic Contents of

E. tectorius Leaf and Fruit Extracts

The total polyphenolic contents (phenolics, tannins, and flavonoids) of different extracts of the E. tectorius leaf were analyzed and presented in Table 17.3. In this study, the successive ethyl acetate leaf extract of E. tectorius (658.66 ± 26.06 mg gallic acid equivalents (GAE)/g of extract) shows a higher total phenolic content, while the methanol leaf extract (48 ± 4.45 mg GAE/g of extract) has a higher tannin content. However, the flavonoid content was found to be higher in the water leaf extract (226.25 ± 5.12 mg RE/g of extract). The petroleum ether leaf extract showed the lower presence of the total polyphenolic contents (phenolics, tannins, and flavonoids).

From the results, it was observed that high polar and middle polar extracts contain more polyphenolic contents than non-polar extracts, because water, methanol, and ethyl acetate solvent extracts are higher in polarity than non-polar solvents like dichloromethane and petroleum ether. Hence, there was a close correlation between the bioactivity and the amount of phenolics, tannins, and flavonoids present in the plants, and this plays a significant role in important biological functions. The polyphenolics from medicinal plants have been reported to have multiple biological properties, including anti-oxidant, anti-diabetic, and anti-inflammatory properties. Recent studies suggest that diets rich in polyphenolics play an important role against oxidative stress related disorders because of their anti-oxidant activities (Murugan and Parimelazhagan 2014; Saravanan and Parimelazhagan 2014; Sathyanarayanan et al. 2017; Muniyandi et al. 2017). Therefore, the polyphenolics (phenolic, tannin, and flavonoid) of E. tectorius may have the properties to react to oxidative stress related disorders.

TABLE 17.3

Determination of Total Phenolics, Tannins, and Flavonoids Content in E. tectorius

Sample

Extracts

Total Phenolics (mg GAE/g extract)

Tannins (mg GAE/g extract)

Total Flavonoids (mg RE/g extract)

Leaf

Petroleum ether

41.80 ±6.54d

25.33 ± 1.89'

29.300 ± 0.63'

Dichloromethane

26.73 ± 4.63'

30.53 ± 3.62"

93.050 ± 3.78'

Ethyl acetate

658.66 ± 26.06“

42.80 ± 7.62“

117.50 ±3.00b

Methanol

324.00 ± 29.22b

48.00 ± 4.45“

70.13 ± 1.201

Water

133.73 ±7.06'

41.86 ± 13.81“

226.25 ± 5.12“

Note: Values are mean of triplicate determination (n = 3) ± SD. GAE: gallic acid equivalents, RE: rutin equivalents. Statistically significant at p < 0.05 where •>b>c>d>e in each column.

The numbers in bold indicate that the result is highly significant.

17.3.4 In Vitro Anti-oxidant Assays

The free radical scavenging activity of different extracts of the E. tectorius leaf was analyzed and is shown in Table 17.4. By analyzing all the extracts, the ethyl acetate extract of the leaf (9.43 pg/mL) showed better Half Maximal Inhibitory Concentration (IC50) values in the DPPH radical scavenging activity. The minimal activity was seen in the dichloro- methane leaf extract (148.21 (Xg/mL). The lower IC50 value indicates the higher DPPH radical scavenging activity. The DPPH radical has been used widely to evaluate the antioxidant activity of the plant extracts and foods as free radical scavengers or hydrogen donors (Soare et al. 1997). The highly enhanced DPPH radical scavenging activity of the ethyl acetate extract was observed in the present study. It may be due to the polar nature of the solvent and the extracting ability of the polyphenolics from the E. tectorius leaf that was studied.

The ferric reducing ability of the E. tectorius extracts was estimated based on their ability to reduce a TPTZ-Fe (III) complex to TPTZ-Fe (II). The ethyl acetate extracts of the leaf (8066.66 mM' (Fe (II) E/mg extract) had the highest ferric reducing power, followed by the methanol leaf extract. However, compared to all the solvent extracts, the petroleum ether extract of the E. tectorius leaf showed the minimal scavenging activity. Thus, the ferric reducing power of different extracts of E. tectorius revealed that there are phytocompounds in the ethyl acetate leaf extract which have a high affinity to the ferric ions, and thereby quench them through redox reactions. The reducing power of the bioactive phytocompounds, mainly the high molecular polyphenolics, was associated with anti-oxidant activity, specifically scavenging of free radicals (Su et al. 1987; Siddhuraju and Manian 2007). Results have revealed that the E. tectorius extracts with the higher amount of phenolics have higher reducing power.

The leaf of the E. tectorius was analyzed to determine the total anti-oxidant potential by a phosphomolybdenum assay. Among the different extracts used, the ethyl acetate extract of the leaf (1460.66 mg AAE/g extract) showed the higher reducing ability of Mo (VI) to Mo (V) and the subsequent formation of the green phosphate/Mo (V) complex at an acid pH (Murugan and Parimelazhagan 2014; Thangaraj 2016). The lowest reducing ability was seen in the petroleum ether extract of E. tectorius. Thus, the total anti-oxidant capacity observed for the extracts of

E. tectorius can be correlated with its free radical scavenging activity w'hich is equivalent to that of natural antioxidant ascorbic acid. The reduction of Mo (VI) to Mo (V) by the extracts of the E. tectorius leaf may be due to the electron transfer or hydrogen ion transfer by the bioactive phytocompounds, specifically the polyphenolics present in the leaf. From the results, the higher radical scavenging activity revealed by the E. tectorius leaf extracts points out that the plant is highly potential, and the extracts can also scavenge other free radicals in the body.

17.3.5 Anti-microbial Activity

The in vitro anti-microbial activity against the UTI-responsible pathogenic strains E. coli, S. aureus, P. aeruginosa, and C. albicans was examined for successive petroleum ether, dichloromethane, ethyl acetate, methanol, and w'ater extracts from the E. tectorius leaf. The anti-microbial activities of these extracts were evaluated by the well diffusion method. The diameter of the inhibitory zones recorded includes the size of the well (6 mm in diameter). The anti-microbial activity of the E. tectorius extracts on the different organisms causing UTIs is given in Table 17.5.

The present study indicated the great variation in the antimicrobial activities of the E. tectorius leaf extracts against gram-positive, gram-negative, and yeast urinary tract pathogens. The maximum anti-bacterial and anti-candidal activity was shown by the ethyl acetate and methanol extracts of the

E. tectorius leaf with the inhibitions of a 21.00 ± 1.00 mm to 13.66 ± 0.28 mm zone size at 1 mg concentration (Figure 17.1). Similar results were reported by fruit extracts against the urinary tract pathogens (Manoharan et al. 2019). The ethyl acetate extracts of the E. tectorius leaf were found to be active against E. coli (15.66 ± 0.57 mm Zone of Inhibition

TABLE 17.4

Determination of Phosphomolybdenum and FRAP Assay of E. tectorius

Sample

Extracts

DPPH Radical Scavenging Activity IC50 Value (gg/mL)

FRAP mM (Fe(ll)E/g extract)

Phosphomolybdenun (mg AAE/g extract)

Leaf

Petroleum ether

75.29“

165.78 ± 11.14“

160.00 ±9.75'

Dichloromethane

148.21'

180.19 ±23.11“

335.16 ± 14.33“

Ethyl acetate

9.43"

8066.66 ±887.15“

1460.66 ± 278.68"

Methanol

20.66b

3717.64 ± 326.82b

912.33 ± 196.00b

Water

63.89'

1021.56 ±315.80'

377.00 ± 21.79'

Rutin (control)

10.83

4705.69 ± 27.66

312.22 ±30.97

Note: Values are mean of triplicate determination (n = 3) ± SD. AAE: ascorbic acid equivalents. Statistically significant at p < 0.05 where a>b>‘>d>' in each column.

The numbers in bold indicate that the result is highly significant.

Antimicrobial activity of Elaeocarpus tectorius leaf extracts 1

FIGURE 17.1 Antimicrobial activity of Elaeocarpus tectorius leaf extracts 1. Petroleum ether leaf extract; 2. dichloromethane leaf extract; 3. ethyl acetate leaf extract; 4. methanol leaf extract; 5. water leaf extract; 6. 4% DMSO control; and 7. positive control (Bacteria-Ampicillin/Candida - Miconazole).

TABLE 17.5

Antimicrobial Activity of E. tectorius Leaf Extracts

Extracts

S. aureus

E. coli

P aeruginosa

C. albicans

ZOIa (mm)

MIC

MBC (Mg mL)

ZOIa (mm)

MIC (Mg rnL)

MBC (Mg mL)

ZOIa (mm)

MIC (Mg rnL)

MBC (Mg mL)

ZOIa (mm)

MIC (Mg mL)

MFC

(Mg/mL)

Petroleum ether

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

07.33 ± 0.57

450

550

Dichloromethane

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

Ethyl acetate

16.83 ± 0.76

125

150

15.66 ± 0.57

150

250

15.41 ± 0.52

200

250

18.33 ± 1.52

200

350

Methanol

15.58 ±0.38

150

200

13.66 ±0.57

200

400

13.66 ±0.28

300

350

21.00 ± 1.00

150

250

Water

12.33 ±0.28

150

200

12.83 ±0.28

200

400

09.50 ± 0.50

400

500

17.16 ± 1.04

250

350

4% DMSO

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

00.00 ± 0.00

-

-

Ampicillin (15 pg)

22.16 ±2.56

0.6

1.0

19.00 ± 1.73

0.8

1.0

15.50 ±0.50

0.7

1.0

NA

NA

NA

Miconazole (30 pg)

NA

NA

NA

NA

NA

NA

NA

NA

NA

22.00 ± 1.00

1.00

1.50

* Data are presented in mean values (n = 3) ± Standard deviation at l mg concentration of extracts. ZOI: Zone of inhibition, MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal concentration, NA: Not Applicable.

The numbers in bold indicate that the result is highly significant.

(ZOI), 150 (Jig mL MIC, 250 pg/mL MBC), S. aureus (16.83 ± 0.76 mm ZOI. 125 |ig/mL MIC. 150 pg/mL MBC), P. aeruginosa (15.41 ± 0.52 mm ZOI, 200 pg/mL MIC, 250 pg/ mL MBC), and C. albicans (18.33 ± 1.52 mm ZOI, 200 pg/ mL MIC, 350 pg/mL MFC), whereas the methanol leaf extract of E. tectorius showed major activity with 21.00 ± 1.00 mm of clear inhibition zone, 150 pg/mL MIC, and 250 pg/mL MFC against C. albicans. The petroleum ether leaf extract has shown the least activity against only C. albicans, but other tested bacteria showed no resistance (Table 17.5). Previous reports from India have revealed that the ethanol extract of Zingiber officinale, Punica granatum, Terminalia chebula, Ocimum sanctum, Cinnamomum cassia, and Azadirachta indica possessed good anti-microbial activity against UTI pathogens (Sharma et al. 2009).

Worldwide, microbial infections are one of the most serious issues in the twenty-first century. The appearance of microbial resistance to antibiotic drugs is a most important health problem, and so it is essential to develop new antibiotic drugs with new mechanisms of action against multidrug resistant microbial strains to defeat these problems (Morris and Masterton 2002; Shaikh et al. 2015; Elangomathavan et al. 2015). Plants have usually provided a source of hope for novel phyto-compounds, as poly herbal mixtures have made great contributions to human health care. The use of plants and their products wfith knowm anti-microbial activities can be of great significance for therapeutic treatments (Kumar et al. 2012).

More than 95% of UTIs are caused by E. coli, w'hich is the most commonly infecting bacterial pathogen, and others only 5%. About 35% of healthy women suffer the symptoms of a urinary tract infection, and about 5% of women every year suffer with the problem of painful urination (dysuria) (Kumar et al. 2012). UTIs caused by a yeast fungal species are the second most common cause of nosocomial UTIs in children and women. It can spread systemically and can be life threatening. The occurrence of UTIs due to the Candida species increased gradually w'ith a prevalence rate 27.2% (Sefton 2000). Plants and their products have been used for several years to control different UTIs. They can affect the UTI pathogens as disinfectants, analgesics, and diuretics (Ubaid et al. 2016). The active components of the plant extracts may display their microbicide potential either by degradation of the cell wall, disruption of the cytoplasmic membrane, affect the synthesis of DNA and RNA, hold up the enzymatic activities inside the cell, and affect nutrient uptake and electron transport (Shan et al. 2007).

From the above results of anti-microbial activity, the ethyl acetate and methanol extracts of the E. tectorius leaf extract exhibited broad-spectrum activity against tested UTI pathogens as compared to the petroleum ether and dichlorometh- ane extracts. The susceptibility of the UTI pathogens varied for polar solvent extracts and non-polar solvent extracts. This indicates the contribution of more than one active principle of biological significance for botanicals (Ming et al. 2005). The traditional healers use mainly water as the solvent, but we observed that extracts prepared from the E. tectorius leaf in ethyl acetate, methanol, and water as solvents provided a more reliable anti-microbial activity against target pathogens (Parekh 2007). In the present investigation, negligible inhibitory activity was observed in the E. tectorius leaf petroleum ether and dichloromethane extracts, which may lack the solubility of the active phyto-constituents. This study may explain the use of E. tectorius by indigenous people against various infections for generations. The E. tectorius extracts studied here had shown that they are potentially rich in anti-microbial chemical substances and suggest that the extracts may be used for treatment of urinary tract infections.

17.3.6 UV-Vis Spectral Measurement

The ultraviolet/visible spectroscopy can be used for the qualitative analysis of plant extracts because the aromatic molecules in plant extracts are powerful chromophores under ultraviolet/visible range. Natural polyphenolics can be determined by using UV-visible spectroscopy. The phenolic compounds including flavonoids, tannins, anthocyanins, polymer dyes, and phenols form complexes with iron that have been detected by the UV-Vis spectroscopy (Kemp 1991). The UV-Vis absorbance spectra of the ethyl acetate and methanol leaf extract of the E. tectorius were recorded in the range of 250-800 nm and have spectral shapes and intensities (Figure 17.2). The methanol E. tectorius leaf extract exhibited a high absorbance intensity in the range of 250-350 nm with a maximum absorbance

at 278 nm. The absorbance in the range of between 250 and 350 nm is associated with the n-n electronic transitions and usually comes from aromatic phytocompounds, hydroxyl, carbonyl, and other chromophores. These phyto-compounds are natural phenols, and they have poor solubility in water at acidic and physiological pH. Therefore, some natural phenolics are not suitable for use as drugs (Tomren et al. 2007). The UV-Vis spectra of the E. tectorius ethyl acetate leaf extract is very different with the E. tectorius methanol extract because of the high absorbance intensity in the range of 250-750 nm with a maximum absorbance at 411 nm. In this wavelength, the rc-7t electronic transitions are responsible for displaying the absorption of the UV-Vis radiation (Kim et al. 2013). These transitions are due to the presence of a high amount of polyphenolics (total phenolic extract (280 nm), flavones (320 nm), phenolic acids (360 nm), flavonoids (510 nm), the total anthokyanids (520 nm) and tannins (710 nm), and other secondary metabolite compounds present in the plant leaf extract (Urbano et al. 2006). From this spectral analysis of both extracts, the spectral region in the range of 250-350 nm will be very useful to discriminate the E. tectorius methanol extract from the ethyl acetate extract. Therefore, the spectroscopic UV-Vis analysis gives clear differentiation and information on the composition of the total polyphenolics content and other phyto-compounds present in the successive ethyl acetate and methanol leaf extracts of the E. tectorius.

This present study showed that the extraction is an essential step to find active phytocompounds with biological properties from plant extracts. Comparison of these solvent extracts from the E. tectorius leaf by successive extraction methods revealed that they produce dissimilar results; however, there are quantitative differences in the total polyphenolics content, anti-oxidant, and anti-microbial activities. Overall, successive ethyl acetate extraction was found to be the best in obtaining anti-oxidant and anti-microbial chemical substances from the E. tectorius leaf.

17.3.7 TLC Separation

The phyto-constituents of the extracts were separated by TLC analysis. The TLC profiling of ethyl acetate and methanol extracts of E. tectorius gives a distinguished result that expresses the presence of a number of phyto-compounds. The ethyl acetate extract of E. tectorius harbors more phytocompounds, followed by the methanol extract (Figure 17.2). Similar kinds of phyto-compounds were identified from both extracts with various concentrations based on the recovery percentage of the organic solvents used in successive extraction. The major phytochemical constituents were identified from both extracts of E. tectorius by UV-visible spectroscopy and thin layer chromatography analysis. These studies revealed that the E. tectorius extracts have highly significant anti-oxidant and anti-microbial compounds and are useful in the treatments of several diseases. This information will help in the selection of a suitable solvent system for the further separation of phytocompounds from the E. tectorius extracts. The medicinal significance of the plant is due to the fact that each of the bioactive compounds generates characteristic physiological actions on humans (Jagadeesan et al. 2019). It is obvious that the leaf extracts of E. tectorius contains various phyto-compounds which may have the appropriate biological properties to be used in the pharmaceutical industry.

17.4 CONCLUSION

This investigation has been done in order to evaluate the effects of different solvents on the successive extraction of bioactive compounds from the E. tectorius leaf and the in vitro antioxidant activities and anti-microbial effects against target UTI pathogens. The results showed that the polarity of different solvents affect the total phenolics content and anti-oxidant activities of the E. tectorius leaf extracts and the ethyl acetate extract exhibited the greatest polyphenolics content, antioxidant and anti-microbial activities, followed by the methanol extract. It can be concluded that the ethyl acetate was the most effective solvent for the extraction of soluble phenolic constituents from the E. tectorius leaf. The significant antimicrobial activity against UTI pathogenic bacteria and fungi demonstrated the anti-microbial potency of the E. tectorius leaf. In conclusion, the E. tectorius leaf contains potential anti-oxidant and anti-microbial phyto-constituents that may be of great use in the pharmaceutical and food industries as a remedy against UTIs and other infectious diseases. Further detailed studies are required for the isolation and identification of botanicals from the E. tectorius leaf related to anti-oxidant and anti-microbial activity.

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