Exploring the Synergistic potential of Polyherbal Extracts in Enhancing Antimicrobial and Antioxidant Activities

Phytochemicals, primarily derived from plant sources, are bioactive compounds classified as secondary metabolites. The economic significance of secondary metabolites is evident in their applications across various industries, including pharmaceuticals, flavourings, fragrances, dyes, pigments, pesticides, and food additives. Many pharmaceuticals are either synthetic modifications or direct replicas of these naturally occurring substances (Twinkle S et al., 2015).

Traditional medicinal plants, rich in phytochemical constituents, are widely used in formulations for skin-related ailments, particularly for wound healing (Bharadhan Bose et al., 2024).  Herbal remedies have shown potential in treating conditions such as ulcers, wound infections, skin diseases, inflammation, scabies, leprosy, and venereal diseases (Akshay Sharma et al., 2021).

 Antioxidants play a crucial role in enhancing quality of life by preventing or delaying the onset of degenerative diseases. Oxidative stress, a concept widely studied in medical sciences over the past three decades, is actively involved in the pathology of common diseases such as diabetes, hypertension, preeclampsia, atherosclerosis, acute renal failure, Alzheimer’s disease, and Parkinson’s disease (Nur Alam et al., 2013). During oxygen metabolism, cells generate reactive oxygen species (ROS), which are potentially harmful. Antioxidants neutralize free radicals, preventing their negative impact on living organisms. In particular, antioxidants play a vital role in counteracting oxidative stress. (Irina Georgiana et al., 2021).

The concept of polyherbal formulations is well established and has achieved remarkable success in modern medicine, offering new hope to patients. In the pharmaceutical industry, several studies have demonstrated that combination therapy using plant extracts and antibiotics is more effective against diseases such as diabetes and cancer than monotherapy (Patel and Saravolatz, 2006). Polyherbal formulations were found to be more effective than individual constituents (Abd El-Kalek and Mohamed, 2012; Mundy et al., 2016).

PLANT DESCRIPTION:

MATERIALS AND METHOD:

COLLECTION OF PLANT SAMPLES

The leaves of the plant samples (Morinda pubescens, Terminalia catappa, and Tridax procumbens) were collected from the local market region of Ganapathy, Coimbatore district Tamil nadu India. The plant material was identified and its authenticity confirmed by comparing it with the voucher specimen at the herbarium of the Botanical Survey of India, Southern Circle, Coimbatore, India.

SAMPLE PREPARATION

The collected sample (Morinda pubescens, Terminalia catappa, and Tridax procumbens) were washed with a running tap water the plant material was dried by shaded dry for four days. The plant materials were coarsely powdered separately. By using a grinding miller to form fine solid. Somnath et al.,2018

PREPARATION OF EXTRACT

The extraction process is done with the ethanol. To take 2g of each plant dried powder sample it treated with the 20ml ethanol. The mixture was allowed to stand for 24hours in a room temperature. The extract was then filtered through a Whatman filter paper. The extraction stored for further activities. Jaya patal et al., 2023

PREPARATION OF POLY HERBAL EXTRACTS

Polyherbal extract was prepared by taking 3 g of extract of all plants, that is, Morinda pubescens Terminalia catappa, Tridax procumbens and properly mixing to get 180 ml of polyherbal extract stored for further analysis.

QUALITATIVE PHYTOCHEMICAL ANALYSIS 

Phytochemical screening of the polyherbal extracts was assessed by standard methods Tiwan et al., 2011 Swamini kute 2024

• Test for alkaloids - Wager’s test
• Test for flavonoids - Alkaline reagent test
• Test for Reducing sugar test – Fehling’s test
• Test for Saponins - Foam test
• Test for phenol - Ferric chloride test 
• Test for tannin - Ferric chloride test
• Test for terpenoids - Salkowki test
• Test for steroids - Salkowki test
• Test for protein - Ninhydrin test
• Test for glycosides

ANTIOXIDANT ACTIVITY

DPPH radical scavenging activity

Antioxidant activity was assessed using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical inhibition assay. Several concentrations of compounds (10 to 50 µl) were prepared for the sample Solution. To each sample solution 500 µl of DPPH (100 µg/mL) and 0.5mL of ethanol were added. The mixture was incubated for 30 min. The absorbance was measured at a wavelength of 517 nm Kumar et al., 2011. A control was prepared wing 0.5 ml of the respective vehicle in place of filtrate or ascorbic acid. 80% ethanol was taken as a hank. (Mahdi-Pour et al.,2012). A plant extract with IC 50 <100 µg/ml has a strong antioxidant activity. While one with IC 50 50mg/ml is considered a very strong antioxidant agent. (Jumina et al., 2019)

FRAP

The antioxidant activity of the plant extracts is determined using FRAP method. In a test tube, aliquots Several concentrations of compounds (10 to 50 µl) were prepared for the sample were added respectively. 3.8ml of FRAP reagent were added. This reagent was previously prepared by mixing 10 parts of 300mM sodium acetate buffer solution at pH 3.6. 1 part of 10mM TPTZ and 1 part of 20mM FeCl2 hexahydrate. The resulting mix was incubated for 30 minutes at 37?C. The absorbance increase was measured at 593nm in a UV spectrophotometer. A control was prepared using 0.5ml of respective vehicle in place of filtrate or ascorbic acid. Blank reacted with distilled water.  (Chaves et al., 2020). A plant extract with IC 50<100µg/ml has a strong antioxidant activity. While one with IC 50<50mg/ml is considered a very strong antioxidant (Jumina et al.,2019).

ANTI MICROBIAL ACTIVITY

Antibacterial Activity

Agar Well Diffusion method

 The antibacterial activity of polyherbal extract was determined by Well Diffusion method (Bauer et al., 1996). The 25 to 100 μl of extract was poured into the wells. After that, the plates were incubated at 37°C for 24 hours. Assay was carried into triplicates and control plates were also maintained. Zone of inhibition was measured from the edge of the well to the zone in mm. The tested cell suspension was spread on potato dextrose agar. Well were put into the agar medium using sterile forceps. Plant extract were poured on to wells. Then plates were incubated at 37°c for about 24 hours and control was also maintained. Zone of inhibition was measured from the clear zone in mm.

Agar well diffusion method has been used to determine the antimicrobial activities and minimum inhibitory concentrations or plant extracts against Gram-positive, Gram-negative bacteria.  The extracts exhibited antibacterial activities against tested microorganisms.

RESULT AND DISCUSSION

COLLECTION OF PLANT SAMPLE

The leaves of the plants Morinda pubescens, Terminalia catappa, Tridax procumbent (L) were collected from the local market in the region of Ganapathy Coimbatore district               

Morinda pubescens

Terminalia catappa              Tridax procumbent

SAMPLE PREPARATION

The samples were washed with distilled water and dried away from the sunlight. The dried samples were powdered.

PREPARATION OF EXTRACT

Ethanol is used for the sample extraction for 24 hours at room temperature using the maceration method. And the extracted sample was filtered. The extract was stored in a container in refrigerator for further use                               

PREPARATION OF POLY HERBAL EXTRACTS

Polyherbal extract was prepared by the ratio of 1:20 where the 3g of the each plant sample of Morinda pubescens, Terminalia catappa, Tridax procumbent (L) in the 180ml of the ethanol as solvent.

QUALITATIVE PHYTOCHEMICAL ANALYSIS 

The polyherbal extract of Morinda pubescens, Terminalia catappa, Tridax rocumbent (L) were subjected to various qualitative phytochemical tests to determine the phytochemical constituents present in the extract

+++” indicates strongly positive, “++” indicates positive, “-” indicates negative

ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS

Ethanolic extract of Morinda Pubescens  was found to be contains flavonoids, Phenols, reducing sugar, tannin in higher content. Steroids, alkaloids, terpenoids, Saponin were found to be present in leaf extract.

Ethanolic extract of Terminalia catappa was found to be contains alkaloids, flavonoids, Phenols, Saponins, Reducing sugar, tannin in higher content. Protein, steroids, terpenoids found to be present in leaves extract.

Ethanolic extract of Tridax procumbent was found to be contains flavonoids, Phenols, reducing sugar, tannin in higher content. Saponins, steroids and terpenoids found to be present in leaf extract. Polyherbal leaves extract of Morinda Pubescens, Terminalia catappa ,Tridax procumbent(L) was found  to be contains the alkaloids, flavonoids, Phenols, reducing sugar,  tannin In the higher content steroids, terpenoids are present. So the polyherbal leaf extract has been contains the most of the bio active component

EVALUATION OF ANTIMICROBIAL ACTIVITY:

Evaluation of DPPH

The DPPH radical scavaging activity of polyherbal extract were investigated. Ascorbic acid was used as a standard at concentration (10 – 50 μl/ml) it showed inhibitory activity from 15% to 84%. In the polyherbal extract at concentration (10-50μl) showed inhibitory activity from 36% to 71% respectively

In In vitro antioxidant activity of polyherbal extract is observed at different concentrations. The result shows DPPH activity of the polyherbal extract and compared with each other with standard Ascorbic acid. It was found that polyherbal extract had the higher activity than the standard Ascorbic acid

Evaluation of FRAP assay

The FRAP radical scavenging activity of polyherbal extract were investigated. Ascorbic acid was used as a standard at concentration (10 – 50μl/ml) it showed inhibitory activity from 30% to 81%. In the ethanolic poly herbal extract at concentration (10-50μl) showed inhibitory activity from 60% to 80% respectively

The In vitro antioxidant activity of polyherbal extract is observed at different concentrations. The result shows FRAP activity of the polyherbal extract and compared with each other with standard Ascorbic acid. It was found that poly herbal extract had the similar activity

EVALUATION OF ANTI MICROBIAL ACTIVITY:

Agar well diffusion method

The antibacterial activity of ethanolic extract of Morinda pubescens, Terminalia catappa, Tridax procumben was found good against gram positive bacteria and gram negative bacteria shows the zone of inhibition against in both Escherichia coli and Staphylococcus aureus. The concentration of the sample was taken as 25,50,75,100 µl of respectively. The highest antibacterial activity is seen at the highest concentration of 100 µl of the sample. The activity that is zone of inhibition increases with the increase in concentration of sample, shows that ethanolic extract of Morinda pubescens, Terminalia catappa, Tridax procumben potent antibacterial activity against Staphylococcus aureus and Escherichia coli, when compared with standard.

The agar diffusion a test of the antibiotic sensitivity of bacteria. It is widely used to determine the zone of inhibition of bacteria. Bacteria which are used to identify antimicrobial activity are Escherichia coli, Streptococcus aureus

E.coli                       S.aureus