long, slender fruit with rounded edges to its surface[3]. It is employed in conventional medicine to combat bacterial illnesses. Helicteresisora is useful and curing for hypolipidaemic, anti-oxidant, hepatoprotective, and cardiotonic actions, according to research by Sharma and Chaudhary (2016)[4] . Additionally, it has been noted that the root juice of Helicteresisora is useful in treating fever (induced by Salmonella bacteria), asthma (caused by bacteria that cause Mycoplasma pneumonia and MRSA pneumonia), cough (caused by Bordetella pertussis)[5].

Helicteresisora has phytochemical components such Glyceriods like amygdalin, Rosmarinic Acid, phlorotannins, and saponins, which are vital for plants' antibacterial activity. Additional non-water soluble phytochemical substances, such as alkaloids and anthraquinones in the aqueous extract of Helicteresisora, were also found in another study (Kumar et al. 2007 Heading Abstract)[6].

Piper longum and Helicteresisora had different effects on gram-positive and gram-negative bacteria[7,8]. Some possible mechanisms to inhibit bacterial growth by phytochemicals are interacting with membrane protein and disintegrating the cell membrane, coagulating cell content, inhibiting DNA gyrase, and inhibiting protein biosynthesis[9].

Staphylococcus epidermidis causes inflammation, sinus infection and wound infection. My grandfather has been a victim of sinus infection, and most of sinus tissue have been badly damaged. It is a gram-positive bacterium with a thicker peptidoglycan cell wall.

Escherichia coli  is a Gram-negative bacteria with thin cell walls of peptidoglycan and an outer membrane with lipopoly saccharide  and causes Stomach pain, Vomiting, Diarrhea, etc[10]. Stomach problems have been common in my family, thus it encouraged me to explore this bacteria.

My personal career goal is to become a biotechnologist, and our class experiments inspired me to measure the herbs' inhibitory zone against gram-positive and gram-negative bacteria in order to learn more about their biological importance. The measurement of the inhibitory zone's diameter was done using a dependable, conventional technique. Furthermore, the Soxhlet (hot) extraction method is precise in extracting antibacterial phytochemicals and the extraction is performed in aqueous solution. It is a continuous process utilizing the principle of siphoning and reflux and requires less time than another extraction process like maceration and percolation. It produces higher extract concentration with better-quality extract without changing the chemical properties of active constituents. Five concentrations of plant extract would be enough to produce reliable results. The higher the concentration, the more antibacterial components would be present, leading to a faster reaction rate (killing of bacteria). This would give a relatively large inhibition zone diameter and vice versa. Other extraction methods like reflux extraction or decoction can be used, but they consume more time and have some water-soluble impurities[11].

2.Literature review

The findings of the research conducted by Mahajan and Itankar 2020[12] confirm this view. Helicteres isora differed significantly in its ability to inhibit gram-positive and gram-negative bacteria's development, and that it had a greater impact on gram-positive stain depending on the kind of extract used in the experiment. Compared to aqueous extract and other organic solvent extracts, ethanolic extract had the largest variation in the zone of inhibition, indicating that the extraction solvent has an impact on the assessed antibacterial activity. In contrast, Tambekar et al.'s (2008) research demonstrates that an aqueous extract exhibited the largest inhibitory zone and the highest yield, indicating that the extract selection was accurate. Gram-positive bacteria were equally or more affected by Helicteresisora. Accepting the null hypothesis, it may be said that there was no discernible difference between the gram-positive and gram-negative bacteria in this Helicteresisora experiment. High plant extract concentration values require more investigation because they appear to have a stronger impact on gramme negative bacteria compared to gramme positive bacteria, which runs counter to every finding in the literature review.

3. Method

A.     Hot Extraction method (For the extraction of leaves)

Gather 60±0.01 gram of each type of plant. Use a mortar and pestle to crush, and a mixer grinder to grind into a fine powder. Put 50±0.01g of leaf powder in 170±0.5ml of petroleum ether to defat it. To prevent the liquid from evaporating, pack it snugly and cover it with cling wrap. Spread the treated powder on the paper to allow the petroleum ether to evaporate after a full day. Apply Whatman filter paper to the porous thimble to prepare it. Weigh out 15± 0.01g of powder in the porous bag, then set it within the device's main chamber. Fill the flask with 200±0.5 ml of distilled water.  Connect the extractor's tubes carrying the cold water supply and the condenser. 

The moment at which the colour of the solution in the thimble becomes entirely transparent at 90°C indicates that the plant extract has been extracted fully after around 16 hours of heating. Using a spatula, transfer the distillation flask solution into the beaker. To obtain dry extract, use a water bath set at 80°C to evaporate the extraction liquid. Put a label on it.

B.     Stock solution preparation

To create 100 mg/ml of stock solution, add 1± 0.01g of extract to 10±0.1ml of distilled water in a small bottle and mix gently. Fold the foil paper over the tiny bottle. Put the bottle in the sonicator to get the extract dissolved.

C.     Antibacterial Testing

Well Diffusion Method and inhibition zone Measurements

a.      Well Diffusion Method

Transfer the Agar media to each agar plate evenly and leave it under laminar air flow for 2 hours to settle. Take the Sterile Swab, put it into the bacterial culture, and then rub the swab in the agar plate (use bacterial culture, which is in stationary phase, to have maximum and equal bacterial population). Create the well using the sterile borer (sterilized using Flame) diameter of - 6mm. Using a micropipette, add 40±0.8 µl of 5 Different Concentrations of extract. (Change the Micro-tip after every trial). Seal the plates partly and Put them in the incubator for 72 hours at 25°C. After 72 hours, measure inhibition zone (Diameter) using inhibition zone measuring scale.

b.      Iinhibition zone Measurements

Small white circular bacteria colonies were visible after the incubation. Some inhibition zone had dark yellowish color for circles, and others had light circular yellowish color.

The color change was visible in solvent in soxhlet extraction before and after plant extraction. The color from transparent changed to dark black color for both plants

Fig. 3 Inhibition zone for Staphylococcus epidermidis for trial four against Helicteresisora

4.Results and discussion

Effect of increasing concentration of Helicteresisora on inhibition zone measurement in mm (± 0.5mm) against Staphylococcus epidermidis and Escherichia coli showed in Table 1.

Table 1: Effect of increasing concentration of in gram positive and gram negative bacteria.

Graph 1 shows logarithm curve for Average inhibition zone for Helicteresisora against Staphylococcus epidermidis and Escherichia coli (Error bars are standard deviation)

Table 3 shows the rate change (gradient) for Helicteresisora from graph 1 calculated by GDC

The graph displays a logarithmic increase in the Helicteresisora zone of inhibition, with an initial sharp gradient and a later less steep gradient. The rate change decreases with increasing extract concentration. At higher extract concentrations, rate change is very low (Table 3) as the zone of inhibition is reaching plateau, suggesting a saturation point (Maximum strength of extract).

The graph showed Helicteresisora produces greater inhibition zone against Staphylococcus epidermidis than against Escherichia coli at lower extract concentrations. At higher concentrations greater inhibition zone was produced against Escherichia coli than against Staphylococcus epidermidis. Thus, as plant extract concentration increases, greater inhibition zone is produced.

The same inhibition zone value for both bacteria by Helicteresisora is observed in graph 1 at the intersection point of the graph.

The error barsin the graph represent the standard deviations that mostly overlap, giving insignificant differences.This is because Gram-positive bacteria are easy to kill as they have porous peptidoglycan layer, allowing easy phytochemical component’s penetration. However, gram-negative bacteria have additional impermeable outer membraneswhich resist antibacterial components at lower concentrations to enter bacteria.

Moreover, the poorly water-soluble components like alkaloids, glycoside, and flavonoids, when diluted with water, wouldbe present in very less quantity, leading to lower value of inhibition zone on Escherichia coliat low concentration. The control value is not included in logarithmic curve as the curve does not passes origin (0,0). The total mass obtained of Helicteres isora after extraction was 5.01±0.01gm

Effect of increasing concentration of Piper longum on inhibition zone measurement in mm (± 0.5mm) against Staphylococcus epidermidis and Escherichia coli shown in Table 4

Table 4: Effect of increasing concentration of Piper lingum in Gram positive and Gram negative becteria.

Graph 2 shows logarithm curve for average inhibition zone value for Piper longum against Staphylococcus epidermidis and Escherichia coli

Table 5 shows the rate change for Piper longum from graph 1 calculated by GDC

The rate change (table 5) for the zone of inhibition zone for Escherichia coli was consistently higher than that of Staphylococcus epidermidis, indicating a logarithmic rise in the zone of inhibition by Piper longum with a steep gradient early but a less steep gradient later.

In graph two, at high concentrations, Piper longum is more effective at Escherichia coli than Staphylococcus epidermidis.

The literature value of P.D. Lokhande et al. 2007[13] suggests that Piper longum has an equal or more significant effect on gram-positive bacteria than on gram-negative bacteria. The error bars in Graph 2 do not overlap for lower values. However, theyoverlap at high values. It can be concluded Piper longum shows significantly different result only for lower plant extract concentration, and for high extract concentration, it is insignificant.

It is easy for phytochemicals to penetrate in gram positive than in gram negative bacteria, due to membrane structure. More research needs to be done for high plant extract concentration values, as they show greater effect on gram negative bacteria than on gram positive bacteria which contradicts most literature review, and structural aspects.

The total extract mass obtained of Piper longum was 3.98±0.01gm

Statistical Test –

One way Anova analysis-

Since it is not evident from the graph, this statistical test was used to investigate the statistical difference of the 100 mg/ml extract concentration of Helicteresisora and Piper longum on Staphylococcus epidermidis and Escherichia coli (with reference to graphs 1 and 2). Whether or not the outcome is statistically different at other concentrations is made evident by the overlapping error bars.

Figure 4 Test for Helicteresisora on Staphylococcus epidermidis and Escherichia coli

Figure 5 Test for Piper longum on Staphylococcus epidermidis and Escherichia coli

According to a one-way ANOVA analysis, the F-statistic value for both plants (Figures 4 and 5) has a p-value larger than 0.05 (5% Significance Level), indicating that there is no statistically significant difference in the mean inhibition zone of Helicteresisora against Escherichia coli and Staphylococcus epidermidis. The same is true for Piper longum. Since there aren't many changes between the two microorganisms.

Graph 3 shows antibacterial activity of both plant

5. Conclusion