INTRODUCTION:

Food poisoning resulted due to microbial contamination presents a significant public and economic health problem for human society. Amongst many strategies for inhibiting the growth of undesirable microorganisms, in order to avoid food spoilage and ultimately to poisoning, is the use of chemical agents commonly known as ‘preservatives’.¹ Various kinds of microorganisms such as gram negative bacteria like Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa along with gram positive like Staphylococcus aureus, Bacillus cereus and fungi like Aspergillus niger are common food spoiler.

Most outbreaks of Escherichia coli, rod shaped gram negative bacteria, had been linked with the consumption of undercooked products, pork sausages and salami. The ability of Escherichia coli to adapt to acidic environment in gastro intestinal tract made it as one of the most dangerous pathogens in the food products.^2,3 Aspergillus niger is a fungus responsible for the disease called ‘black mold’ to fruits and vegetables as well as a common food contaminant.⁴While, Bacillus cereus is estimated to be responsible for 1%–12% of all food poisoning outbreaks worldwide with diarrheal and emetic syndrome.^5-13

Although chemical preservatives have proven its efficient role in controlling the growth of such undesired microorganisms, but still, newer ways are required to avoid their unpleasant side effects as well as growing microbial resistances as a serious concern. For such purposes, researchers are focusing the use of plant extracts with antimicrobial activities as preservatives due to its relative safety.¹⁴

Caesalpinia pulcherrima (L.) (Family- Fabaceae) commonly known as ‘peacock flower’ is widely known ornamental tree distributed throughout the tropical and sub-tropical region including India, China, Africa and native to West Indies.^15,16

C. pulcherrima reported to have various pharmacological activities like anthelmintic, antimalarial, antidiabetic and antimicrobial attributed to richness of phytoconstituents like saponins, terpenoids, tannins, alkaloids and many more.^17-20 Various scientific studies have concluded the relative safety of C. pulcherrima leaf extracts with LD₅₀ value of more than 2,000 – 4,000mg/kg in experimental Wistar albino rats and mice respectively.^21,22 Due to all such findings, our main objective of the study was to evaluate the antimicrobial activity of C. pulcherrima leaf extracts against food borne pathogenic and spoilage bacteria and fungi namely Bacillus cereus, Escherichia coli and Aspergillus niger HN-2 respectively.

MATERIALS AND METHODS:

Chemicals and microorganisms:

For this study, Nutrient agar (HiMedia, India), Petroleum ether, Chloroform and Acetone (CDH Fine Chemical, India), Ethanol (Changshu Hongsheng Fine Chemical, China) of AR grade were used. The water used for the extraction was double distilled prepared in the laboratory while for antimicrobial study, the sterile water, prepared in the laboratory, was used. This study was conducted using Bacillus cereus (Gram negative bacteria), Escherichia coli (Gram positive bacteria) and Aspergillus niger HN-2 (Fungus).

Plant Materials:

Caesalpinia pulcherrima (L.) leaves were collected from botanical garden of college at Anjora, Chhattisgarh region (21.19664˚ N, 81.21988˚ E). The taxonomical authentication of said plant material was confirmed from the Department of Botany, Digvijay College, Rajnandgaon, Chhattisgarh. Further, the collected plant material was washed thoroughly with water to remove any adhering dust and undesired materials followed by drying in shade and grinding to fine powder through 100 mm sieve.

Extraction and phytochemical screening:

The powdered plant material was successively extracted with solvents- petroleum ether, chloroform, acetone, ethanol and water using soxhlet apparatus for 7 days and the organic solvents were recovered by using rotary vacuum evaporator. The extracts were further concentrated and dried using water bath and subjected to phytochemical screening for the presence of carbohydrates, proteins, alkaloids, saponins, anthraquinone glycosides, steroids, flavonoids, terpenoids, tannins and amino acids along with antimicrobial study. For the phytochemical screening, various standards procedures were followed.^23-29

Antimicrobial assay and minimum inhibitory concentration (MIC):

The antimicrobial assay was carried out using agar well diffusion method; the plates were incubated at 35-37°C for 24hours (for Bacillus cereus and Escherichia coli) and 96 hours for (Aspergillus niger HN-2).

The minimum inhibitory concentration (MIC) represents the lowest concentration of substance, as an antimicrobial, that can inhibit the visible growth of microorganism after overnight incubation.^30,31 In order to determine the MIC’s of extracts, the initial extracts concentration of 50mg/ml were evaluated for growth inhibition. Overall, for the MIC’s of extracts, the concentrations used were in the range of 10-100mg/ml. The experiment was repeated thrice and diameter of inhibition zone, in mm, by respective extracts was noted down as mean ± standard deviation.

RESULTS AND DISCUSSIONS:

The plant materials used in the current study, was confirmed as Caesalpinia pulcherrima (L.) belonging to family Fabaceae. A herbarium specimen (No: ACP/HER/M.Ph-2009/01/22) was deposited to Department of Botany, Digvijay College, Rajnandgaon, Chhattisgarh along with Pharmacognosy and Phytochemistry Department of Apollo College of Pharmacy, Durg (C.G.) for future reference.

The extractive values (through soxhelation method) were found to be- petroleum ether soluble (0.9±0.1%), chloroform soluble (1.1±0.1%), acetone (3.4±0.3%), ethanol (4.6±0.2%) and water (8.4±1.2% through decoction method). These extractive values clearly indicated the presence of higher polar constituents in the leaves. On phytochemical investigation of various extracts, to determine the classes of phytoconstituents present, the carbohydrates were found present in ethanol as well as in aqueous extracts, proteins and amino acids in aqueous extract, alkaloids in petroleum ether and acetone extracts, saponins in chloroform, ethanol and aqueous extracts, steroids in petroleum ether and chloroform extracts, terpenoids were reported in all extracts except aqueous, tannins and flavonoids in all five extracts while anthraquinone glycosides were absent in all the extracts. The details of phytochemical investigation are given in the table (Table1).

Table 1: Phytochemical screening

Phytoconstituents	Tests	Petroleum ether extract	Chloroform extract	Acetone extract	Ethanol extract	Aqueous extract
Carbohydrates	Molisch’s	-	-	-	+	+
	Fehling’s	-	-	-	+	+
	Barfoed’s	-	-	-	+	+
	Bial’s Orcinol	-	-	-	+	+
	Selwinoff’s	-	-	-	+	+
	Tollen’s phloroglucinol	-	-	-	+	+
Proteins	Biuret	-	-	-	-	+
	Million’s	-	-	-	-	+
	Sulphur containing	-	-	-	-	+
Alkaloids	Mayer’s	+	-	+	-	-
	Wagner’s	+	-	+	-	-
	Hager’s	+	-	+	-	-
	Dragendroff’s	+	-	+	-	-
Saponin glycosides	Foam	-	+	-	+	+
Saponin glycosides	Haemolysis	-	+	-	+	+
Anthraquinone glycosides	Borntrager’s	-	-	-	-	_
Steroids	Salkowski’s	+	+	-	-	-
Steroids	Legal’s	+	+	-	-	-
Flavonoids	Shinoda	+	+	+	+	+
Terpenoids	Salkowaski’s	+	+	+	+	-
Terpenoids	Liebermann-Burchard’s	+	+	+	+	-
Tannins	Ferric chloride	+	+	+	+	+
	Lead acetate	+	+	+	+	+
	Potassium dichromate	+	+	+	+	+
	Gelatin	+	+	+	+	+
Amino acids	Ninhydrin	-	-	-	-	+

+ represents“present”and–represents“absent”

Fig. 1: Antimicrobial assay against (A)- Bacillus cereus, (B)- Escherichia coli and (C)- Aspergillus niger HN-2; Et- Ethanolic extract, Aq- Aqueous extract, Ac- Acetone extract, Ch- Chloroform extract, Pe- Petroleum ether extract and C- Control

The antimicrobial assay clearly indicated that, the petroleum ether and acetone extracts have growth inhibition of at least two microbial strains. Further, Petroleum ether extract of C. pulcherrima showed inhibition of Bacillus cereus (diameter of inhibition zone – 33mm) and Escherichia coli (diameter of inhibition zone – 17mm) with MIC value of 50mg/ml and 80 mg/ml respectively while showed no growth inhibition of Aspergillus niger HN-2 up to 100mg/ml. Similarly, acetone extract showed inhibition of Aspergillus niger HN-2 (diameter of inhibition zone - 38mm) and Escherichia coli (diameter of inhibition zone – 31mm) with MIC value of 40mg/ml while ineffective against Bacillus cereus up to 100mg/ml. Chloroform, ethanol and aqueous extracts were found to show no particular inhibition to all three tested microorganisms up to the concentration of 100mg/ml (Fig.1 and Table 2).

Table 2: Antimicrobial assay and MIC

Extracts	Minimum Inhibitory Concentration (MIC)
Extracts	*Bacillus cereus*	*Escherichia coli*	*Aspergillus niger* *HN-2*
Petroleum ether	50mg/ml	80mg/ml	> 100mg/ml
Chloroform	> 100mg/ml	> 100mg/ml	> 100mg/ml
Acetone	> 100mg/ml	40mg/ml	40mg/ml
Ethanol	> 100mg/ml	> 100mg/ml	> 100mg/ml
Aqueous	> 100mg/ml	> 100mg/ml	> 100mg/ml

CONCLUSION:

Food poisoning and spoilage are common problems nowadays that can threaten the health of humans. Modernization and scientific approach helped us to control such situation by the use of artificial preservatives. But due to increased capacity of microorganisms to resist such preservatives and toxicity chances, we need some better option that can be efficiently effective against them. In search of better options, researchers are continuously studying the use of plant extracts with antimicrobial action as preservatives. In continuation of such explorations, in this study, the antimicrobial activity of C. pulcherrima leaves extracts were evaluated against Bacillus cereus, Escherichia coli and Aspergillus niger HN-2 responsible for most cases of food poisoning and spoilage. The petroleum ether extract was found effective against Bacillus cereus and Escherichia coli while acetone extracts against Aspergillus niger HN-2 and Escherichia coli. This study concluded that, the C. pulcherrima leaves extract can be used as preservatives to control the food poisoning and spoilage.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

ACKNOWLEDGEMENT:

The authors would like to acknowledge Central Instrumentation Facility (CIF) of Apollo College of Pharmacy, Durg, India for the provided instrumental facilities and financial support during course of work.

REFERENCES:

1. Amensour M, Bouhdid S, Fernandez-Lopez J, Idaomar M, Senhaji NS, Abrini J. Antibacterial activity of extracts of Myrtus communis against food-borne pathogenic and spoilage bacteria. Int J Food Prop. 2010; 13(6): 1215-1224.

2. Hakim AS, Abuelnaga ASM, Ezz-Eldeen AM, Bakry MA, Ismail SA. Prevalence of some food poisoning bacteria in local and imported retail pork by-products in Egyptian markets. Afr J Microbiol Res. 2015; 9(22): 1492-1498.

3. Sachidananda Swamy HC, Asha MM, Chaithra M, Vivek MN, Kambar Y, Prashith Kekuda TR. Antibacterial activity of flower extract of Caesalpinia pulcherrima, Delonix regia and Peltaphorum ferrugineum against Urinary tract Pathogens. Int Res J Biological Sci. 2014; 3(4): 80-83.

4. Sharma R. Pathogenecity of Aspergillus niger in plants. Cibtech J Microbiol. 2012; 1: 47-51.

5. Dagenais TR, Keller NP. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clin Microbiol Rev. 2009; 22(3): 447-465.

6. Tewari A, Abdullah S. Bacillus cereus food poisoning: International and Indian perspective. J Food Sci Technol. 2015; 52(5): 2500-2511.

7. Gupta V, Singh S, Gupta YK. Synthesis, characterization and antimicrobial activity of Co(II), Ni(II), Cu(II) and Zn(II) complexes N- O- S donor ligands. Asian J Pharm Ana. 2014; 4(4): 174-177.

8. Killedar SG, More HN. Screening of antimicrobial potential and phytoconsituents for different extracts of Memecylon umbellatum Burm Inflorescences. Asian J Pharm Res. 2011; 1(4): 114-118.

9. Paul S, Saha D. Comparative study of the efficacy of Barleria prionitis leaf extracts against bacteria. Asian J Pharm Res. 2012; 2(3): 107-110.

10. Mohite SA, Shah RR, Patel NR. Antimicrobial activity of leaves extracts of Jatropha curcas. Asian J Pharm Res. 2018; 8(1): 17-20.

11. Mohite S, Shah R, Patel N. Antimicrobial activity of leaves extracts of Passiflora foetida. Asian J Res Pharm Sci. 2018; 8(1): 17-20.

12. Meera R, Devi P, Madhumitha B, Kameswari B. Antibacterial activity of crude extracts and semi synthetic hydrazone derivatives of Rimelia reticulate. Asian J Research Chem. 2009; 2(4): 445-447.

13. Granum PE, Lund T. Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett. 1997; 157(2): 223-228.

14. Mostafa AA, Al-Askar AA, Almaary KS, Dawoud TM, Sholkamy EN, Bakri MM. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Saudi J Biol Sci. 2018; 25(2): 361-366.

15. Lukita B, Budiardjo S, Elya B. Evaluation of the antimicrobial activity of Caesalpinia pulcherrima (L) Swartz extract against microbes that cause dental and oral infections and determination of the total flavonoid and total phenolic contents of the plant. Iran J Pharm Sci. 2019; 15(4): 1-10.

16. Osuntokun OT, Julianah JU, Thonda OA. Bioprospective screening of antibacterial and phytochemical activity of Caesalpinia pulcherrima (pride of Barbados) on selected clinical isolate. Bioequiv and Bioavailab Int J. 2017; 1(3): 000118.

17. Patil N, Vaishnav RL, Thanusubramanian H, Holla SN, Manohar HD, Bairy KL. Formulation and evaluation of the hydroalcoholic extract of Caesalpinia pulcherrima (Stem bark) on wound healing model in wistar rats. Int J Adv Res. 2015; 3(3): 648-654.

18. Sasi Premila JM, Sreeja N. Preliminary phytochemical, antioxidant and antimicrobial analysis of Caesalpinia pulcherrima. World J Pharm Res. 2018; 7(5): 1400-1408.

19. Mallapur S, Biradar PR, Kavatagimath SA, Patil P, Kudatarkar N. Pharmacological evaluation of Caesalpinia pulcherrima leaf extract for anticancer activity against Ehrlich Ascites Carcinoma bearing mice. Indian J Pharm Edu Res. 2021; 55(1): 164-173.

20. Faloye K, Famuyiwa S, Akinwunmi K, Tata C, Ayoola M, Fakola E, Akinyele O, Ndinteh D. Antioxidant potentials of the pod extract of Caesalpinia pulcherrima Swarta (Fabaece) and the theoretical evaluation of the antioxidant property of the isolated compounds. Res Sq. 2021; DOI: 10.21203/rs.3.rs-1137829/v1.

21. Sharma V, Rajani GP. Evaluation of Caesalpinia pulcherrima Linn. for anti-inflammatory and antiulcer activities. Indian J Pharmacol. 2011; 43(2): 168-171.

22. Kumar S, Singh J, Baghotia A, Mehta V, Thakur V, Choudhary M, Verma S, Kumar D. Antifertility potential of the ethanolic extract of Caesalpinia pulcherrima Linn. leaves. Asian Pac J Reprod. 2013; 2(2): 90-92.

23. Kokate CK. Practical Pharmacognosy. Vallabh Prakashan, 2014.

24. Banarase N, Khadabadi S, Sawarkar H. Pharmacognosy practicals (an illustrative guide). Scholar’s Press, 2018.

25. Khandelwal KR, Sethi V. Practical pharmacongosy (techniques and experiments). Nirali Prakashan, 2016.

26. Narasimhan R, Satiyamoorthy M. Phytochemical screening and antioxidant studies in the pulp extracts of Cucurbita maxima. Asian J Pharm Res. 2016; 6(1): 01-04.

27. Roy A, Bhoumik D, Sahu RA, Dwivedi J. Phytochemical screening and antioxidant activity of Sesbania grandiflora leaves extracts. Asian J Res Pharm Sci. 2014; 4(1): 16-21.

28. Morris SS, Jayabalan G, Jha AK, Verma S, Swarnkar Y. Phytochemical screening and in-vitro athelmintic activity of seed extracts of plants Carum carvi of Family Apiaceae. Asian J Res Pharm Sci. 2016; 6(4): 246-254.

29. Mouli JBC, Madhu C. Reddy KR, Lakshmi PJ. Omar Md. Comparative Studies on Phytochemical screening and metal analysis of alcoholic extracts of Musa acuminate, Actinidia deliciosa and Mangifer indica by using ICPOES and Flame Photometer. Asian J Res Pharm Sci. 2019; 9(2): 78-84.

30. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal. 2016; 6(2): 71-79.

31. Sanchez E, Morales CR, Castillo S, Leos-Rivas C, Garcia-Becerraa L, Martinez DMO. Antibacterial and antibiofilm activity of methanolic plant extracts against Nosocomial microorganisms. Evid-based Complement Altern Med. 2016, 2016: 1572697.

Received on 25.02.2023 Modified on 15.09.2023

Res. J. Pharmacognosy and Phytochem. 2024; 16(1):5-8.

DOI: 10.52711/0975-4385.2024.00002