Nature is the potential source of medicinal agents and these agents have been used for thousands of years and numbers of modern drugs have been isolated from natural sources. Various medicinal plants have been used for years in daily life to treat various diseases all over the world. Approxi-mately, amongst 1500 identified medicinal plants 500 are commonly in use (Chidambaram et al., 2014). Plants produce a diverse range of bioactive molecules (Uddin et al., 2017). Higher plants as a major source of medicinal compounds to play a dominant role in the maintenance of human health since ancient times (Farombi et al., 2003). The WHO estimates that 80% of the world population use herbal medicine for some aspects of primary health care purposes (Shinwari et al., 2009). There are 4,22,127 plant species growing on planet earth, about 35,000 to 70,000 plants species are used as medicinal plants (Hasan et al., 2007). The practice of traditional medicine is widespread in China, India, Japan, Pakistan, Sri Lanka and Thailand (Hasan et al., 2007). Calendula is used in ayurveda for the treatment of fever, diarrhea, and cancer (Krag et al., 2013). 

Medicinal plants are one of the necessary and valuable resources in a wide range of natural resources that they can have an important role in health. C. officinalis is globally known for its medicinal importance containing various photo-chemical activities. Knowledge of the various biological activities and chemical constituents of medicinal plants are desirable not only for the discovery of new therapeutic agents but also for information in discovering new sources of other economic materials (Khaleqzzaman et al., 2002). Natural products have been a major source of the new drugs (Vuorelaa et al., 2004). The potential for developing antibacterial into medicine appears rewarding, from both the perspective of drug development and the perspective of phytomedi-cines (Rahaman et al., 2004). It possesses cyto-toxic as well as abnormal cell growth reducing potential. Traditionally, C. officinalis was used as anti-inflammatory, diaphoretic, analgesic, anti-septic and in jaundice treatment (Chakraborthy et al., 2010). It is also used as a mouthwash after tooth extractions (Mukesh et al., 2011).

Phytopharmacological studies of different Calen-dula extracts have shown anti-viral activity, anti-HIV properties of therapeutic interest, and anti-genotoxic properties (Perez-Carreon et al., 2002). Calendula extract-containing creams and gels are commonly used to treat skin irritation, diabetes, inflammation, and burns, especially after radio-therapy, and to aid wound healing (Edwards et al., 2015). C. officinalis is widely cultivated as an herb and can be grown easily in sunny locations in most kinds of soils. C. officinalis are considered by many gardening experts as one of the most versatile flowers to grow in a garden, especially since they are easy to grow, and tolerate most soils. Pot marigolds typically bloom quickly from seed (in under two months) in bright yellows, golds, and oranges. Some scientists indicate the high anti-microbial activity of Calendula stems (Goyal et al., 2011). C. officinalis was very effective in inhibit-ing the growth of the zone of inhibition E. coli and B. subtilis of Petroleum ether and chloroform was found in cm respectively (Md et al., 2014). However, gm negative organisms are more sen-sitive to the extract of Calendula. It may be used as a constituent of a drug (Happy et al., 2018).  The aim of the study was to identify bio-active chemical compounds from the flower of C. officinalis, their antimicrobial activity and setting up the standards specification.

 Collection, Processing and Preservation of Calendula officinalis plant material - Healthy, disease free, mature C. officinalis plants was identified and selected for the collection of leaves from the garden of Kushtia (23°5437.1"N 89°07 20.9"E) and Jhenidah (23°3240.1"N 89°1027.8"E) Pouroshova, Bangladesh. After cleaning the waste materials of the leaf then plant material was air dried in room temperature. After 7 days the dried plant was grinded to form fine powder from the blender machine. This powder was used for the preparation of different solvents extracts by sequential extraction. The Good Agricultural and Field Collection Practices (GACP) of medicinal plants of World Health Organization (WHO) were followed strictly.

Solvents and Chemical used - In this experiment, only a few selected solvents are used such as petroleum ether and chloroform. Petroleum ether, chloroform, etc are used in this experiment.

Disk Preparation - The filter paper was punched with the punching machine and disc was made. The disc paper was taken into the test tubes & sterilized in an autoclave for 15 minutes with 15 psi and 121 ºC temperatures.

Medium preparation - In this study, nutrient agar medium was used for antibacterial screening. For the test 2.8gm of the nutrient agar media was taken into 500 ml autoclave conical flask. The media properly dissolved with the distilled water then sterilized in an autoclaved for 15minutes with 121ºC. After autoclaving, the media was cooled for some time and poured into the autoclaved Petri dishes in the laminar airflow cabinet.

Inoculum preparation - 1ml of distilled water was taken into the screw-capped tube and the pure colony of freshly cultured bacteria was added into the tube and vortexes. The OD was measured with the colorimeter and microbial population was confirmed to be within tube. This suspension was used as inoculums.

Determination of minimum inhibitory concen-tration (MIC) of Calendula officinalis - MIC of the most active chloroform and petroleum ether extracts were determined using serial dilutions of 512mg/ml, 512µg/ml to 2µg/ml in chloroform and petroleum ether solvent against both strains of E. coli in Agar well diffusion method as mentioned earlier. The lowest concentration of the extract required to inhibit the growth of the organism in vitro is MIC. In the present study, it was deter-mined following the serial dilution technique.

Preparation of sample solution - Stock working solution of the plant extracts were prepared by dissolving 10gm of the dried extracts in 1ml each of petroleum ether and chloroform solvent into two separate flasks. From these solutions, 1ml solution was added to 9ml petroleum ether. Then from this 2nd solution 8532µl was added to 1468µl chlo-roform. Therefore, the final concentration was reached to 512µg/ml. From these solutions, the 1 ml solution was added to 9ml petroleum ether. Then from this 2nd solution 5688µl was added to 4312µl petroleum ether. Therefore, the final concentration was reached to 512µg/ml. 6hours extraction of 5.12grams seed powder was added to the 10ml chloroform and petroleum ether; there-fore get mother solutions which were used without dilutions.

Serial dilution - For preparing 512µg/ml to 2µg/ml, 1ml of solvent was added to each of nine screw-capped test tubes. 1ml of the sample having 512µg/ml extracts was added to the first test tube containing 1ml of respective solvent and mixed well in the vortex and then 1ml of this solvent was transferred to the second test tube containing 1ml of the same solvent. After mixing well, 1ml of this mixture was transferred to the third test tube. This process of serial dilution was continued up to the last test tube. Finally, the concentration of the last test tube was 2µg/ml.

Preparation of disc - The disc paper was soaked with each concentration of extracts and placed at room temperature for air dry for 15 hours. Then dried disc paper was placed in the oven for 1 hour at 37 ºC. After completion of oven dry, the disc paper was labeled according to different concentration and finally, the labeled disc paper was taken into the vial and it was ready for antibacterial activity.

Antimicrobial activity test - All strains used in the study were inoculated to nutrient agar and incubated at 35±0.1oC for 24h and were allowed to grow until they reach 108-109cfu/ml. Antibacterial activity studies were carried out for each test strains in duplicate and average measurement was calculated. Four organisms, (three gm-negative i.e. E. coli, Salmonella spp, Shigella spp, and Staphy-lococcus spp) were tested in this study to determine the antibacterial effect of crude extracts (Petroleum ether and Chloroform) C. officinalis. 

Antibacterial activity of extracts and crude of C. officinalis was observed. Then in vitro antibacterial activities of the extracts were measured by employing standard agar disc diffusion method. Discs were impregnated with each treatment and control was assayed on duplicate agar medium plate for E. coli, Shigella spp, Staphylococcus spp, and Salmonella spp. The experiment was repli-cated two times to confirm the reproducible results. Sterile, blank paper discs were impregnated with only sterile solvent (Petroleum ether and Chlo-roform) and used as negative control each time. Standard Kanamycin (30µg) and Strepto-mycin (5µg) were used as a positive control for com-parison of the antibacterial activity.

Statistical analysis - The Statistical analysis was performed by using SPSS software (release 10.0) to find out significant differences in the antibacterial effects.

Petroleum ether extract of Calendula officinalis - Petroleum ether extract of C. officinalis exhibit antibacterial activity against B. subtilis, E. coil, S. lutea, K. pneumoniae. Different concentration of petroleum ether extract of Calendula (512 mg/ml) produced a zone of inhibition 1.7cm against B. subtilis, 1.4cm against E. coil, 1.6cm against S. lutea,1.5 cm against K. pneumoniae (Table 1). 

Another concentration of petroleum ether extract of Calendula (512µg/ml) produced a zone of inhibition 1.3cm against B. subtilis, 1.1cm against E. coil, 1.3cm against S. lutea, 1.2cm against K. pneumoniae (Table 1). And another concentration of petroleum ether extract of Calendula (256 µg/ml) produced a zone of inhibition 1.0cm against B. subtilis, 1.0cm against E. coil, 1.2cm against S. lutea, 1.0cm against K. pneumoniae (Table 1). 

Similarly, concentration of petroleum ether extract of Calendula (128µg/ml) produced a zone of inhibition 0.9cm against B. subtilis, 1.0cm against E. coil, 1.0cm against S. lutea, 1.0cm against K. pneumoniae (Table 1).

The concentration of petroleum ether extract of C. officinalis (64µg/ml) produced a zone of inhi-bition 0.8cm against B. subtilis, 0.9cm against E. coil, 0.8cm against S. lutea, 0.9cm against K. pneumoniae (Table 1). The concentration of petroleum ether extract of Calendula (32µg/ml) produced zone of inhibition 0.6cm against B. subtilis, 0.9cm against E. coil, 0.6cm against S. lutea, 0.8cm against K. pneumoniae (Table 1). 

The concentration of petroleum ether extract of Calendula (16µg/ml) produced zone of inhibition 0.6 cm against B. subtilis, 0.8cm against E. coil, 0.5cm against S. lutea, 0.7cm against K. pneu-moniae (Table 1).

The concentration of petroleum ether extract of Calendula (8µg/ml) produced zone of inhibition 0.4cm against B. subtilis, 0.4cm against E. coil, no zone against S. lutea and 0.7cm against K. pneumoniae (Table 1). 

Table 1: Comparison of antibacterial activity and MIC values of leaf extract of C. officinalis in petro-leum ether by inhibition zone.

The concentration of petroleum ether extract of Calendula (4µg/ml) produced a zone of inhibition only 0.7cm against K. pneumoniae (Table 1). In addition, the MIC value was also (0.3cm) determined. The MIC values against K. pneu-moniae were 4ugml-1.

Fig 1: Comparison of Antibacterial activity and MIC values of leaf extract of C. officinalis in petroleum ether by inhibition zone.

Photochemical analysis of Solvents and Chemical - Photochemical analysis showed that almost all the solvents are responsible for extract-ing different components from the plants. Plant pharmacological studies have suggested that Calendula extracts have anti-viral, anti-geno-toxic and anti-inflammatory properties (Eva Jimenez-Medina et al., 2006).

The Chloroform extract of Calendula offi-cinalis - Chloroform extract of C. officinalis exhibit antibacterial activity against B. subtilis, E. coil, S. lutea, K. pneumoniae. Different concen-tration of chloroform extract of Calendula (512 mg/ml) produced a zone of inhibition 1.7 cm against B. subtilis, 1.8cm against E. coil, 1.6 cm against S. lutea, 1.7cm against K. pneumoniae (Table 2). 

Table 2: Comparison of antibacterial activity and MIC values of leaf extract of C. officinalis in chloroform by inhibition zone. 

Another concentration of chloroform extract of Calendula (512µg/ml) produced zone of inhi-bition 1.5cm against B. subtilis, 1.6cm against E. coil, 1.4cm against S. lutea, 1.5cm against K. pneumoniae (Table 2). And another concentration of chloroform extract of Calendula (256µg/ml) produced a zone of inhibition 1.2cm against B. subtilis, 1.2cm against E. coil, 1.2cm against S. lutea, 1.3cm against K. pneumoniae (Table 2). The concentration of chloroform extract of Calendula (128µg/ml) Produced zone of inhi-bition 0.8cm against B. subtilis, 0.9cm against E. coil, 1.0cm against S. lutea, 1.1cm against K. pneumoniae (Table 2).

The concentration of chloroform extract of Calendula (64µg/ml) produced zone of inhibition 0.7cm against B. subtilis, 0.7cm against E. coil, 0.8cm against S. lutea, 0.8cm against K. pneu-moniae (Table 2). The concentration of chlo-roform extract of Calendula (16µg/ml) produced zone of inhibition 0.0cm against B. subtilis, 0.0cm against E. coil, 0.6cm against S. lutea, 0.5cm against K. pneumoniae (Table 2). 

The concentration of chloroform extract of Calendula (8µg/ml) produced zone of inhibition 0.5cm against S. lutea (Table 2). The concen-tration of chloroform extract of Calendula (32µg/ml) produced zone of inhibition 0.0cm against B. subtilis, 0.0cm against E. coil, 0.6cm against S. lutea, 0.7cm against K. pneumoniae (Table 2). The concentration of chloroform extract of Calendula (4µg/ml) produced zone of inhibition 0.5cm only against S. lutea (Table 2). 

Fig 2: Comparison of Antibacterial activity and MIC values of leaf extract of C. officinalis in chloroform by inhibition zone.

In addition, the MIC value was also (0.5cm) deter-mined. The MIC values against S. lutea were 4 ugml-1. For the comparison of the plant extracts activity positive control (different type of antibiotic disc) and negative control (only solvent absorbing disc) was used. The negative control showed no activity against all tested bacteria. The positive control showed significant antibacterial activity against all bacteria. 

The petroleum ether and chloroform extracts of C. officinalis showed the highest antibacterial activity against E. coli and K. pneumonia. In the present study, C. officinalis was very effective in inhibiting the growth of E. coil the zone of inhibition of petroleum ether and chloroform was found. The inhibition of the effect of C. offi-cinalis on E. coli was less than that of to Neo-mycin (30ug) and also investigated that mini-mum inhibitory concentration 2µg/ml of leaf extract of C. officinalis in chloroform against K. pneumoniae and E. coli and in petroleum ether against E. coli.  

The largest inhibitory zone is created by 512 mg/ml chloroform extract against E. coli. The extract of C. officinalis has been reported to possess antibacterial activity; however, gram-negative bacteria are more susceptible to the action of the oil, whereas gram-negative organ-isms are more sensitive of the leaves extract (Ali and Blunden et al., 2003). 

The beneficial health effects of extracts from many types of plants that have been used for years in daily life to treat diseases all over the world. In the present study, the extract of leaves displayed a variable degree of anti-micro-bial activity on different microorganisms. E. coli and K. pneumoniae were found to be more sensitive strain than the others. On the other hand, B. subtilis and S. lutea were found to be more resistant bacteria against the C. officinal-lis leaves examining findings, the widest inhi-bition zone was formed E. coli and K. pneu-monia around. The least inhibitory effects were observed for E. coli. The petroleum ether and chloroform extracts of Calendula showed anti-bacterial activity against E. coli and B. subtilis. In conclusion, this result indicated that extracts of the C. officinalis leaves which were prepared using petroleum ether and chloroform have a strong inhibitory activity on some pathogens. The purpose was to examine the inhibitory effects of C. officinalis leaves extract, some bacteria causing poisoning and harmful for humans. So, the further microbiological investi-gation was confined only on petroleum ether and chloroform fraction and also investigation is necessary to confirm the bioactive principles of the C. officinalis in Bangladesh.  

The research project was financially supported with proper guidance and help for data analysis in the Dept. of Biotechnology and Genetic Engin-eering, Islamic University, Bangladesh. Many thanks, to the co-workers supporting for success-ful completion of the research work.

The authors declared no potential conflicts of the interest with respect to the research, authorship and/or publication of this article.