Crude Flavonoids extracted from crushed seeds of dried ripe fruits of
Microbial infections are one of the important health problems faced by the humanity despite availability of many antimicrobial agents. Hence, there is a continuous effort by scientists in order to identify new antimicrobial agents that help to treat the infectious disease. The need for newer antibiotic sources has increased as the occurrence of multidrug resistant microorganisms has increased. From time immemorial, plants continue to be the most important source of antimicrobial agents as ethnopharmacologically a range of plants are used against different infections. Plants comprise a variety of secondary metabolites, the most important of which are tannins, alkaloids, flavonoids, steroids, and other compounds. As a result, science has stretched its wings to investigate traditional and folk medical agents in order to extract therapeutic benefits from these approaches and discover unique and potent antimicrobial agents for the benefit of humanity.
Dental caries, often known as tooth decay, is a prevalent chronic disease that affects people across the world. Dental caries is the decay of teeth caused by generation of acid in the oral cavity as a result of bacterial colonies in the mouth that ferment and digest carbohydrates. Tooth decay is a serious condition that causes excruciating pain, damages the tooth and its surrounding areas. It also has a negative impact on one's quality of life. Streptococcal species like Streptococcus mutans, as well as some acid-forming facultative anaerobes such as
In view of the above facts, we intended to test the antibacterial potency of the fruit extract of
The plant specimen was collected from the ripe berries of
The chemicals and reagents used for extraction and detection of constituents of the dried ripe fruit seeds of
The seeds of dried ripe fruits of
10 g of the crude seed extract was dissolved in 10 ml of water : ethanol (50:50) mixture and used for the identification of the phytoconstituents. This solution was labelled as test solution.
To 1ml of the test solution, a few drops of Fehling’s A & B reagent was added and boiled for a few minutes. Occurrence of red colour indicated the presence of carbohydrates.
To 3ml of the test sample, a few drops of Wagner’s reagent was added and kept aside for few minutes. Formation of brownish precipitate indicated the presence of alkaloids.
1ml of test sample was combined with pieces of magnesium ribbon and concentrated HCl. After a few minutes, appearance of pink tint indicated the presence of flavonoids.
To 1 ml of the solution, 0.5 ml of 1% lead acetate solution was added. The mixture is shaken and kept aside for a few minutes. Formation of dark bluish black colour indicated the presence of Tannins and Phenols.
To extract the flavonoids, 50 gm of powdered plant material was placed in 1 litre of ethanol and refluxed for 24 hours. The crude extract was filtered and concentrated by evaporating the solvent in a rotary evaporator.
Pathogenic microorganism selected for the study included
Using the disc diffusion experiment, the minimum inhibitory concentration (MIC) of the seed extract that showed antibacterial activity against test pathogens was determined. The MIC was studied using the HIMEDIA M210 medium. The extract was diluted nine times in Thioglycollate broth to determine the MIC. 20 microliters of extract was added to 380 microliters of Thioglycollate broth in the first tube. Dilutions were made by adding 200 microliters of Thioglycollate broth to each of the next 9 tubes sequentially. Then 200 microliter was transferred from the first tube to the first tube containing 200 microliter of Thioglycollate broth. This was regarded as a dilution of 10-1. To create a 10-2 dilution, 200 microliter was transferred from the 10-1 diluted tube to the second tube. The serial dilutions were performed up to a 10-9 dilution. 5 microliter was collected from the needed organisms' stock cultures and added to 2 ml of Thioglycollate broth. 200 microliters of the aforesaid culture suspension were added to each serially diluted tube. The tubes were cultured in an anaerobic jar at 37°C for 48-72 hours and then checked for turbidity.
The first 3 or 5 tubes from the MIC dilutions tubes were plated (which was sensitive in MIC) and incubated for 24 hours, after which the colony count was obtained the next day. MBC is used to determine whether the extract (Drug) has a bactericostatic or bactericidal impact on the organism. If there is no growth, the impact is bactericidal. If there is growth, the bacteriostatic effect is at work. Tubes were incubated in a CO2 Jar at 37°C for 48-72 hours for facultative anaerobes. Tubes were cultured in anaerobic jars for 48-72 hours for strict anaerobes.
After mixing an equal amount of broth with the organism and chemical, it was immediately plated, and the time was recorded as 0 hours. Tubes were stored in the CO2 jar until a later time slot was available. It was cultured or plated every 5 minutes, 10 minutes, 30 minutes, and 2 hours, and incubated according to the growth requirements, i.e., in CO2 and anaerobic jars. The plates were removed after 48-72 hours of incubation and the colony count was recorded.
The crude extract from the powdered seed of dried ripe berries of
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1. |
Carbohydrates |
Fehling’s A & B |
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2. |
Alkaloids |
Wagner’s reagent |
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3. |
Flavonoids |
Mg ribbons + Conc. HCl |
+++ |
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4. |
Tannins and Phenols |
1% lead acetate solution |
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(+++) Good amount, (++) Appreciable amount, (+) Small amount
After identification of phytoconstituents, the dried berries were subjected to extraction of flavonoids for the study of antimicrobial activity. The method employed in the extraction of flavonoids was percolation method using ethanol. An opaque translucent powder of group of flavonoids weighing about 63 g was obtained.
The antimicrobial activity of the seed extract from
MIC activity against
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S |
S |
S |
R |
R |
R |
R |
R |
R |
R |
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S |
S |
R |
R |
R |
R |
R |
R |
R |
R |
*S = Sensitive; R = Resistant,
The Minimum Bacterial Concentration (MBC) was studied against
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1. |
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NG |
NG |
NG |
46 |
52 |
60 |
86 |
92 |
116 |
120 |
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2. |
A a |
NG |
NG |
12 |
18 |
20 |
21 |
30 |
36 |
42 |
50 |
*NG = No Growth,
The range of MIC and MBC of the extract was recorded as 25-100µg/mL for
The Time Kill data of
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110 |
98 |
80 |
34 |
NG |
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A a |
81 |
70 |
41 |
12 |
NG |
*NG = No Growth,
Date of
The crude extract derived from the powder of the seeds of dried ripe berries of
Using the ‘Disc Diffusion Assay,’ the antimicrobial activity of the
The MIC activity against
The MIC and MBC of the extract were found to be between 25 µg/mL and 100 µg/mL both for
In the present study we extracted flavonoids from the seeds of dried ripe fruits of S