G. Mallika ^{1, 2} and K. Shailaja^1*

¹Department of Botany, UCS, Osmania University, Hyderabad Telangana, India.

²Departmne of Botany, Govt. Degree College for Women, Sangaredd, Telangana State, India.

Corresponding Author E-mail: gootymallika@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/2813

Abstract

Objective: The objective of the present investigation is to study the phytochemical composition, antioxidant and antibacterial activity of petroleum ether, chloroform, ethanol, methanol and water extracts of the leaf part of Celtis timorensis Spr. The preliminary phytochemical studies were conducted according to standard procedures. Total phenolic content was estimated using the FCA reagent method. The antioxidant efficiency of extracts was evaluated by using molybdate and DPPH methods. The antibacterial potency of leaf extracts was studied using the disc diffusion method against eight human pathogenic bacterial strains. Results: The results of preliminary phytochemical study revealed the presence of alkaloids, phytosterols, phenolic components, tannins, flavonoids, terpenoids, glycosides and saponins. The total phenolic content of the tested extracts exhibited a range between 8.82 to 68.32 mg GAE/g dwt. The highest total phenolic content was observed in the methanol extract (68.32±1.03 mg GAE/g dwt.) and the highest total antioxidant capacity was observed in the methanol extract of leaf part (700.0±0.71 mg ASE/g dwt.).Regarding DPPH scavenging activity the highest DPPH-reducing activity (>90%) was observed by methanol, ethanol and water extracts of the leaf part. Ethanol and water extracts of leaf samples strongly inhibited the gram-negative bacterial species Pseudomonas aeruginosa and Salmonella enterica (13 mm for each species) respectively. While gram-positive species i.e. Bacillus megatherium Artherobacter protophormiae and P. aeruginosa were moderately inhibited by chloroform, ethanol and water extracts (12 mm for each) respectively. Conclusion: In conclusion, the selected medicinal plant C. timorensis extracts exhibited good antioxidant activity, strong antibacterial activity and rich bioactive components. It required further studies on the isolation, and characterization of active principle to evaluate its pharmacological properties.

Keywords

Antibacterial activity; Celtis timorensis; DPPH; total phenolic content

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Copy the following to cite this article: Mallika G. Shailaja K. Phytochemical Composition, Antioxidant and Antibacterial Studies on Celtis Timorensis Leaf Extract. Biomed Pharmacol J 2023;16(4).

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Introduction

Infectious diseases caused by bacterial species are a major health issue for global population. The major human infections causing bacterial species are Pseudomonas aeruginosa, E. coli, Proteus vulgaris, Staphylococcus aureus and Bacillus subtilis^1,2.Medicinal plants used in traditional medicinal systems are a major source for the treatment of infectious diseases caused by microorganisms. These plants are rich in a wide variety of chemical constituents such as alkaloids, terpenoids, flavonoids and tannins and essentials have been found as potent antimicrobial agents^3,4. Scientific studies on the antibacterial activity of medicinal plants have reported that traditional medicinal plant extracts/ phytoconstituents have potent antimicrobial activity ^{5, 6, 7}.

Celtis timorensis Spr. is a medium-sized tree of flowering plant belongs to the family Ulmaceae, distributed in India, Nepal, Thailand, Vietnam, and Malaysia. Different parts i.e. leaf, stem, root, fruit, etc. of this plant have been used by the tribes of Indian states to cure various human diseases such as dysentery, jaundice, memory enhancement, toothache, urinary tract infection and food^{8, 9, 10, 11}.  The review on phytochemical composition, antibacterial, antioxidant and pharmacological studies on Celtis timorensis indicate that very few and sporadic attempts are noticed on preliminary phytochemical ¹², DPPH¹³ antibacterial ¹⁴, antidepressant¹⁵, acute and sub-acute toxicity¹² and wound healing activity¹⁶of leaf extracts of Celtis timorensis. But no previous report was noticed on phytochemical analysis, molybdate dependant antioxidant, DPPH and antibacterial activity of Celtis timorensisleaf part. Hence, we selected Celtis timorensis leaf extracts to evaluate the phytochemical analysis and antioxidant and antibacterial studies of five extracts.

Materials and methods

Plant material

Celtis timorensis leaves were collected from Tirumala hills of Andhra Pradesh. The plant specimen was identified (Voucher #1379) by Dr K. Madhava Chetty, Assistant Professor (Rtd.), Department of Botany, S.V. University, Tirupati.

Preparation of plant extracts

The collected plant sample was washed with distilled water and dried at room temperature. Fifty grams of plant material was pounded and successively extracted with Petroleum ether (PE), chloroform (CE), ethanol (EE) and methanol (ME) using Soxhlet apparatus for 6-8 hours. The extracts were filtered and concentrated under reduced pressure to dryness and the extracts were used for the assay.  The yield of each extract was depicted (Table 1).

Table 1: Yield of Celtis timorensis leaf extracts

S. No.	Extract Type	Yield (%)
Leaf Extracts
1	Petroleum ether	4.28%
2	Chloroform	2.86%
3	Ethanol	14.32%
4	Methanol	14.56%
5	Water	5.0%

Water extract preparation

One gram of plant material after the successive extraction was taken and soaked in 50 ml distilled water for 24 h and filtered. The filtrate was concentrated in a water bath at 40⁰C, and subjected to phytochemical, antioxidant and antibacterial studies.

Preliminary phytochemical screening

Qualitative phytochemical tests for alkaloids, saponins, phytosterols¹⁷, starch, glycosides, phenolic components, gums and tannins¹⁸were determined using standard methods.

Total phenolic content of Celtis timorensis leaf extracts

The total phenolic quantity of Celtis timorensis leaf extract was estimated using methods as described ¹⁹.

Total antioxidant capacity of Celtis timorensis leaf  extracts

Ammonium molybdate dependant antioxidant capacity of Celtis timorensis leaf extracts was determined using methods as described²⁰.

DPPH reducing activity

DPPH scavenging capacity of Celtis timorensis leaf extracts was determined using methods as described ²⁰.

Antibacterial studies

Microorganisms used

The microorganisms used in the present study are Micrococcus luteus (MTCC 9341), Arthrobacter protopharmiae (MTCC 688), Alkaligenes faecalis (MTCC 10757), Enterococcus faecalis (MTCC 439),  Bacillus megatherium (MTCC 5981), Lactobacillus acidophilus (MTCC 10307), Salmonella enterica (MTCC 3858) and Pseudomonas aeruginosa (MTCC 1688) to test the extracts. The organisms were purchased from the MTCC center, IMTECH, Chandigarh, India.

Antimicrobial activity

The antibacterial efficacy of C. timorensis leaf petroleum ether (PE), chloroform (CE), ethanol (EE) methanol (ME) and water extracts were studied using the disc diffusion method²¹.  Paper discs (4 mm) impregnated with distinct concentrations of plant extracts were placed on the petri-plates, containing 20ml of Mueller Hinton Agar (MHA) media (Peptone 17.5 g, meat extract 2 g, starch 1.5 g and agar-agar 17 g/L) seeded with 0.1ml of overnight grown microbial suspension. The discs saturated with methanol ethyl acetate and petroleum ether served as negative controls. Bacterial-free zones present around the discs were treated as positive results. The zones were measured after 24 hours and tabulated.

Results& Discussion

Preliminary phytochemical screening of medicinal plant extracts is essential to know the phytochemical components present in them. Qualitative phytochemical analysis of petroleum ether, chloroform, ethanol, methanol and water extracts C. timorensis leaf showed the presence of alkaloids, phytosterols, phenolic components, tannins, flavonoids, terpenoids, glycosides and saponins (Table 2).Prasanth Kumar et al., (2014)¹² reported the preliminary phytochemical composition of C. timorensis leaf ethanol extract, revealing the presence of tannins, flavonoids, alkaloids, triterpenoids, saponins, glycosides and carbohydrates.

The total phenolic content of petroleum ether, chloroform, ethanol, methanol and water extracts of C. timorensis leaf was estimated using Folin Ciocalteau reagent method. Gallic acid was used as the standard component and the amount of total phenolic components were expressed in milligrams Gallic acid equivalents per gram dry weight (mg GAE/g dwt.).  The results revealed that the total phenolic content of the tested extracts exhibited a range between 8.82 to 68.32 mg GAE/g dwt. (Figure 1). The highest total phenolic content was observed in methanol extract (68.32±1.03 mg GAE/g dwt.). This may be due to the solubility of phenolic components in methanol. Higher amounts of the total phenolic content of methanol extract of different medicinal plant parts were reported and stated that methanol solvent is efficient for extraction of a good amount of phenolic components ^{22, 23, 24}.

Type of component	PE	CE	EE	ME	WE
Alkaloids	NR	NR	NR	+	NR
Saponins	NR	NR	NR	NR	+
Phytosterols	+	NR	+	+	+
Phenolic components	NR	NR	+	+	+
Tannins	NR	+	+	+	NR
Flavonoids	NR	+	NR	NR	NR
Terpenoids	+	+	+	+	NR
Glycosides	+	+	+	+	NR
Gums and mucilages	NR	NR	NR	NR	NR

PE: Petroleum ether extract; CE: Chloroform extract; EE: Ethanol extract; ME: Methanol extract; WE: Water extract; NR: No Reaction

Figure 1: Total Phenolic content of Celtis timorensis leaf extracts Click here to view Figure

Figure 1 Description: Total phenolic content of C. timorensis leaf extracts was estimated using FCA reagent method. The total phenolic content was expressed in Gallic acid equivalents milligrams/ gram dry weight (GAE mg/g dwt.).

The total phenolic content of leaf water extract of C. timorensis was reported as 9.7 mg ²⁵. In the present study, we found a higher amount of total phenolic content at 31.99 mg GAE/g dwt. The variation in the chemical composition of medicinal plants may be influenced by environmental factors and geographical conditions of the plants growing, collection of plant samples, drying and the part that is used²⁶, ²⁷. In addition to the total phenolic content of water extract, we found the total phenolic content in petroleum ether, chloroform, ethanol and methanol extracts of C. timorensis leaf sample.

The results on the ammonium molybdate-dependant antioxidant capacity of C. timorensis leaf extracts ranged from 174.75±0.29 to 700.0±0.71 mg ASE/g dwt. (Figure 2). The highest total antioxidant capacity was observed in the methanol extract of the leaf part (700.0±0.71 mg ASE/g dwt.). The strong antioxidant capacity of methanol extract may be the presence of a higher amount of phenolic components. The strong antioxidant capacity of methanol extract from different medicinal plants was well reported ^{28, 29, 30}.The results were correlated with the phenolic content of leaf extracts. A similar type of correlation of total phenolic content with total antioxidant activity was reported by several researchers ^{31, 32, 33}.

Figure 2: Total antioxidant capacity of Celtis timorensis leaf extracts Click here to view Figure

Figure 2 Description: Total antioxidant capacity of C. timorensis leaf extracts was estimated using ammonium molybdate reagent method. The total antioxidant capacity was expressed in terms of  ascorbic acid equivalents milligams/ gram dry weight (AAE mg/g dwt.).

DPPH scavenging activity of petroleum ether, chloroform, ethanol, methanol and water extracts of C. timorensis leaf part showed concentration dependant DPPH scavenging activity. The highest DPPH-reducing activity (>90%) was observed by methanol, ethanol and water extracts of the leaf part (Table 3).While petroleum ether extract failed to reduce DPPH purple colour. Ethanol and methanol extracts exhibited the lowest IC₅₀ values (50µg/ml). The strong DPPH-reducing activity of ethanol and methanol extracts of the leaf part may be due to the presence of a higher amount of total phenolic content. Several researchers have reported strong DPPH quenching capacity of methanol or ethanol extracts of medicinal plants ^{34, 35, 36, 37}. Rajaneekar et al., (2013a)¹³ reported the DPPH scavenging activity of methanolic extract of leaf sample. It showed maximum inhibition as more than 100% DPPH scavenging activity at 60 µl/ml  (concentration not mentioned). In the present study, the methanol extract of C. timorensis showed 92% as maximum DPPH scavenging activity at 250µg/ml respectively.

The antibacterial activity of petroleum ether, chloroform, ethanol, methanol and water extracts of the leaf part of C. timorensis revealed that all the tested pathogens exhibited concentration dependant antibacterial activity (Tables 4 to 8). Ethanol and water extracts of the leaf sample strongly inhibited the gram-negative bacterial species Pseudomonas aeruginosa and Salmonella enterica (13 mm for each species) respectively (Tables 6 & 8). While gram-positive species i.e. Bacillus megatherium Artherobacter protophormiae and P. aeruginosa were moderately inhibited by chloroform, ethanol and water extracts (12 mm for each) respectively (Tables 5, 6, 8). Reaming tested organisms expressed very feeble antibacterial activity by all the tested extracts. The strong antibacterial activity of ethanol, chloroform and water extracts of the leaf part of C. timorensis may be due to the presence of a good amount of phenolic components. The phenolic components exhibit their antibacterial action in many ways. It can cause morphological changes in bacterial cells i.e. shapes, wrinkles on cell membrane and damage cell membrane in both outer and inner membranes. The mechanism of antibacterial activity of polyphenols is closely related to the chemical nature, and position of hydroxyl and methyl groups^{38, 39, 40}.

Table 3: DPPH scavenging capacity of leaf extracts of Celtis timorensis

(%) DPPH Scavenging capacity
Ethanol Extract (EE)		Methanol Extract (ME)		Water Extract (WE)		Gallic Acid
Concentration (µg/ml)	% DPPH inhibition	Concentration (µg/ml)	% DPPH inhibition	Concentration (µg/ml)	% DPPH inhibition	Concentration (µg/ml)	% DPPH inhibition
12.5	9.86±1.56	12.5	11.22±1.02	100	11.22±1.02	1	16.21±0.02
25	19.39±1.02	25	23.47±3.68	200	27.21±3.12	2	28.02±0.12
*50	50.16±1.56	*50	49.32±1.18	*300	51.70±1.02	4	48.11±1.21
75	68.71±2.57	75	68.03±2.57	400	75.51±1.02	6	60.04±1.02
100	80.13±4.68	100	78.23±2.12	500	91.50±0.59	8	72.00±1.00
125	86.73±1.56	125	84.35±3.86	100	11.22±1.02	10	84.00±1.1
250	91.84±1.02	250	92.18±0.59	200	27.21±3.12	12	92.01±1.12

Table 4: Antibacterial activity of Celtis timorensis leaf Petroleum ether extract

Microorganism	Petroleum Ether extract Concentration (µg/Disc*) Zone of inhibition (mm)
	50*	100*	150*	300*
Micrococcus luteus (MTCC 9341),	5.33±0.57	6.00±1.0	6.33±0.57	6.66±0.57
Arthrobacter protopharmiae (MTCC 688),	5.67±0.76	6.50±0.5	NA	6.83±0.76
Alkaligenes faecalis (MTCC 10757)	6.00±0.5	6.30±1.08	6.67±0.28	7.00±0.5
Enterococcus faecalis (MTCC 439),	7.17±0.28	6.30±1.08	6.50±0.5	5.83±0.76
Bacillus megatherium (MTCC 5981)	6.50±0.86	8.00±0.5	8.17±0.17	9.00±0.5
Lactobacillus acidophilus (MTCC 10307)	NA	7.66±0.76	8.17±0.57	9.17±0.28
Salmonella enterica (MTCC 3858)	5.50±0.5	6.67±0.28	8.17±0.57	9.33±0.28
Pseudomonas aeruginosa (MTCC 1688)	6.17±0.28	5.67±0.28	7.67±0.76	9.33±0.28

• The concentration of extract µg/disc, NA: No Activity

Table 5: Antibacterial activity of Celtis timorensis leaf Chloroform extract.

Microorganism	Chloroform  Extract Concentration (µg/Disc*) Zone of inhibition (mm)
	50*	100*	150*	300*
Micrococcus luteus (MTCC 9341),	7.50±0.5	6.50±1.0	6.67±0.76	8.17±0.57
Arthrobacter protopharmiae (MTCC 688),	5.67±0.76	7.16±0.57	6.67±0.76	7.83±0.57
Alkaligenes faecalis (MTCC 10757)	6.33±0.28	6.83±0.57	7.17±0.57	9.17±0.28
Enterococcus faecalis (MTCC 439),	6.30±1.08	6.83±0.57	7.67±0.76	9.33±0.28
Bacillus megatherium (MTCC 5981)	7.33±0.28	8.33±0.28	9.17±0.28	12.00±0.5
Lactobacillus acidophilus (MTCC 10307)	6.33±0.28	8.17±0.57	8.33±0.28	9.33±0.28
Salmonella enterica (MTCC 3858)	6.50±1.0	7.50±0.5	8.17±0.57	8.33±0.28
Pseudomonas aeruginosa (MTCC 1688)	6.00±0.5	6.83±0.57	7.17±0.57	9.00±0.5

*Concentration of extract µg/disc.

Table 6: Antibacterial activity of Celtis timorensis leaf Ethanol extract

Microorganism	Ethanol Extract Concentration (µg/Disc*) Zone of inhibition (mm)
	50*	100*	150*	300*
Micrococcus luteus (MTCC 9341),	6.50±1.0	6.67±0.76	7.17±0.57	9.17±0.28
Arthrobacter protopharmiae (MTCC 688),	5.67±0.76	7.50±0.5	8.17±0.57	12.33±0.28
Alkaligenes faecalis (MTCC 10757)	5.83±0.57	6.50±1.0	9.17±0.28	11.05±0.5
Enterococcus faecalis (MTCC 439),	6.67±0.76	7.17±0.57	9.17±0.28	10.67±0.28
Bacillus megatherium (MTCC 5981)	5.83±0.57	6.50±1.0	8.17±0.57	11.05±0.5
Lactobacillus acidophilus (MTCC 10307)	6.50±1.0	7.17±0.57	8.33±0.28	10.67±0.28
Salmonella enterica (MTCC 3858)	5.83±0.57	7.50±0.5	9.33±0.28	11.33±0.28
Pseudomonas aeruginosa (MTCC 1688)	6.50±0.5	8.50±0.5	10.67±0.28	13.17±0.28

*Concentration of extract µg/disc.

Table 7: Antibacterial activity of Celtis timorensis leaf methanol extract

Microorganism	Methanol  extract Concentration (µg/Disc) Zone of Inhibition (mm)
	50*	100*	150*	300*
Micrococcus luteus (MTCC 9341),	6.50±1.0	7.17±0.57	6.67±0.76	NA
Arthrobacter protopharmiae (MTCC 688),	5.67±0.76	6.50±1.0	6.83±0.57	7.50±0.5
Alkaligenes faecalis (MTCC 10757)	5.83±0.57	NA	6.67±0.76	8.33±0.28
Enterococcus faecalis (MTCC 439),	5.33±0.57	5.83±0.57	NA	7.50±0.5
Bacillus megatherium (MTCC 5981)	5.50±1.0	6.50±1.0	6.83±0.57	8.17±0.57
Lactobacillus acidophilus (MTCC 10307)	5.67±0.76	6.83±0.57	8.33±0.28	10.67±0.28
Salmonella enterica (MTCC 3858)	5.33±0.57	6.50±1.0	7.17±0.57	6.83±0.57
Pseudomonas aeruginosa (MTCC 1688)	5.33±0.57	5.83±0.57	6.67±0.76	8.17±0.57

*Concentration of extract µg/disc; NA:  No Activity

Table 8: Antibacterial activity of Celtis timorensis leaf water extract

Microorganism	Water extract Concentration (µg/Disc) Zone of Inhibition (mm)
	50*	100*	150*	300*
Micrococcus luteus (MTCC 9341),	5.33±0.57	5.67±0.76	6.50±1.0	7.17±0.57
Arthrobacter protopharmiae (MTCC 688),	NA	5.67±0.76	6.50±1.0	7.17±0.57
Alkaligenes faecalis (MTCC 10757)	5.33±0.57	6.50±1.0	6.83±0.57	9.33±0.28
Enterococcus faecalis (MTCC 439),	5.50±1.0	6.50±1.0	7.50±0.5	9.17±0.28
Bacillus megatherium (MTCC 5981)	5.33±0.57	6.50±1.0	6.83±0.57	9.17±0.28
Lactobacillus acidophilus (MTCC 10307)	5.50±1.0	6.50±1.0	6.83±0.57	7.50±0.5
Salmonella enterica (MTCC 3858)	6.83±0.57	7.50±0.5	11.33±0.28	13.17±0.28
Pseudomonas aeruginosa (MTCC 1688)	7.17±0.57	9.33±0.28	10.67±0.28	12.33±0.28

*Concentration of extract µg/disc; NA:  No Activity

Conclusion

The present study results state that Celtis timorensis leaf extracts were rich in different groups of secondary metabolites. Of the tested five extracts, methanol extract showed a higher amount of total phenolic content and higher antioxidant capacity. Ethanol and methanol extract strongly inhibited DPPH radical in a concentration dependant manner. Ethanol and water extracts of leaf samples strongly inhibited the gram-negative bacterial species Pseudomonas aeruginosa and Salmonella enterica respectively. While gram positive species i.e. Bacillus megatherium Artherobacter protophormiae and P. aeruginosa were moderately inhibited by chloroform, ethanol and water extracts respectively. The strong antibacterial activity of ethanol, chloroform and water extracts of the leaf part of C. timorensismay be due to the presence of a good amount of phenolic components. In conclusion, the selected medicinal plant C. timorensisextractsexhibited good antioxidant activity, strong antibacterial activity and rich bioactive components. It required further studies on the isolation, and characterization of active principle to evaluate its pharmacological properties.

Conflict of interest

No conflict of interest

Funding source

No funding source

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