Natural Remedies for Throat Infections: A Study on the Phytochemistry and Bioactivities of Phyllanthus emblica and Adhatoda vasica

Indigenous medicines have garnered significant attention for their potential as novel antibacterial agents. A wide array of secondary metabolites such as alkaloids, tannins, and flavonoids derived from plant extracts exhibit promising antimicrobial properties, offering hope for new antibiotic development particularly against drug-resistant bacteria [1]. Polyherbal formulations represent a synergistic interaction between multiple herbs. The underlying principle is that phytochemical constituents from one plant may enhance the activity of those from another, potentially improving therapeutic outcomes. This approach also reduces the need for consuming multiple individual herbal preparations [2].Human healthcare continues to be a major global concern, and plant-based therapies collectively known as "phytomedicines"play a vital role in addressing various health issues. These medicines are typically derived from different plant parts such as bark, leaves, flowers, roots, fruits, and seeds. In the present study, two commonly available medicinal plants, Adhatoda vasica and Phyllanthus emblica (Indian gooseberry), were selected for investigation. Phyllanthus emblica is widely used in traditional medicine, with all parts of the plant having therapeutic applications. Its leaves contain important secondary metabolites such as flavonoids, alkaloids, and tannins, which have demonstrated antimicrobial activity against several bacterial pathogens linked to sore throat [3]. Extracts prepared using methanol and acetone solvents showed strong antibacterial activity and possessed additional properties such as anti-inflammatory, antioxidant, anti-cancer, adaptogenic, anti-diabetic, and immunomodulatory effects. Adhatoda vasica, commonly known as Adalodaka, is also a powerful medicinal plant used for treating numerous ailments, including leprosy, skin conditions, piles, cough, and asthma. Its therapeutic potential is attributed to secondary metabolites such as vasicine, vasicinone, vasicine acetate, vasicol, and adhatodine [4]. The plant’s leaves, in particular, have been effective in managing respiratory issues including sore throat, which is a common affliction involving the upper respiratory mucosa. Bacterial sore throat is often caused by pathogens such as Staphylococcus aureus, Klebsiella spp., and Pseudomonas spp., and accounts for approximately 5–10% of sore throat cases. Typically categorized as acute respiratory tract infections (e.g., pharyngitis, sinusitis, or the common cold), they may be accompanied by symptoms such as fever, headache, malaise, and nausea. While antibiotics can reduce symptoms marginally, herbal formulations offer a moderate yet effective alternative. These can be administered as macerations, infusions, decoctions, or tinctures. Often, individuals with sore throat do not seek immediate medical attention. However, untreated bacterial infections can significantly impact daily life by impairing basic functions like swallowing, eating, speaking, and even sleeping. Since bacterial infections account for about 20% of sore throat cases, there is a pressing need for safe, accessible alternatives to conventional antibiotics [5]. The present study explores the pharmacological potential of a synergetic effect composed of Adhatoda vasica and Phyllanthus emblica leaf extracts. The aim is to evaluate their efficacy against common throat infection-causing bacteria and to promote the use of traditional medicines as a viable therapeutic option.

MATERIALS AND METHODS

Collection and processing of plant samples:

The leaf samples of Phyllanthus emblica & Adhatoda vasica were collected from Palakkad, Kerala, India. All the collected plant leaves were washed thoroughly and dried under shade for a few days. The dried plant leaves were powdered and stored in airtight containers for further studies.

Preparation of crude extract:

Acetone was used for the preparation of crude extract, since our preliminary tests showed a better yield compared to other solvents. For extraction, 7.5 g of each dried plant powder was mixed with 150 mL of acetone. The mixture was initially shaken manually and then subjected to continuous agitation using an orbital shaker for 72 hours to ensure maximum extraction of phytochemicals [6]. After the extraction period, the mixture was filtered using Whatman No. 1 filter paper. The filtrate was then subjected to solvent evaporation under reduced pressure or at room temperature until a dry residue was obtained. The resulting crude extract was stored at 4?°C for further analysis.

Collection and Identification of clinical isolates

The samples used in this study were isolated from throat swabs. It was collected from G.H Hospital, Palakkad, Kerala.The cultures were then subjected to gram staining, culturing on selective medium and standard biochemical tests for identification.

Phytochemical Screening:

The presence of bioactive compounds including alkaloids, flavonoids, phenols, tannins etc. present in the plant extracts were identified using standard qualitative a phytochemical screening methods [7] and  Gas Chromatography and Mass spectrometry (GC-MS) analysis

GC –MS analysis

GC –MS analysis of plant extract was performed using the equipment Shimadzu –GCMS. The equipment has a model number: QP20105 with a ELITE-5 MS column with dimensions of 30 meter X 0.25 mm ID X 0.25 micrometer thickness. The identification of components was based on Willey and NIST 11 libraries.(NIST 2011; Willey/NIST 2011) [8,9]. The GC-MS software used was GC-MS Solutions. The instrument was set to an initial temperature of 700C and maintained for 2 minutes. At the end of this period, the oven temperature was raised to 2600C and maintained for 5 minutes. The total GC-MS running time was 51 minutes.

Antibacterial Assay:

The antibacterial activity of the synergistic effect comprising Phyllanthus emblica and Adhatoda vasica was evaluated using the agar disc diffusion method on Mueller-Hinton Agar (MHA) medium.Sterile discs were impregnated with various concentrations of the leaf extract and placed carefully onto the inoculated plates.the stock solution was prepared using 1 mg/mLThe plates were incubated at 37°C for 48 hours. After incubation, the antibacterial activity was assessed by measuring the diameter of the clear zones of inhibition formed around the discs, recorded in millimeters [10].

Antioxidant Activity of extracts by DPPH Free Radical Scavenging Assay:

The antioxidant potential of the synergistic effect of the extract was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging method, as described by Braca et al. (2001) [11]. DPPH is a stable nitrogen-centered free radical that exhibits a deep violet color in solution, which fades to yellow upon reduction through hydrogen or electron donation. Compounds that mediate this reduction are considered potential antioxidants. A range of concentrations of the polyherbal extract (12.5 µg/mL to 200 µg/mL) was prepared from a stock solution, with the final volume of each sample adjusted to 20 µL using DMSO. To each sample, 1.48 mL of DPPH solution (0.1 mM in methanol) was added. A control was prepared using an equivalent amount of distilled water in place of the extract. The reaction mixtures were incubated in the dark at room temperature for 20 minutes. After incubation, absorbance was measured at 517 nm using a UV-Vis spectrophotometer. A solution containing 3 mL of DPPH without any sample served as the control.

The percentage of DPPH radical scavenging activity was calculated using the following equation:

% of Radical scavenging activity = Absorbance of control-Absorbance of sample X 100

       Absorbance of control

RESULTS AND DISCUSSION

The bacterial isolates were confirmed as Klebsiella sps, Pseudomonas sps and Staphylococcus sps. Based on the preliminary biochemical tests. The results were tabulated in Table 1

Table 1: Biochemical characteristics of Clinical isolates from throat samples

Preliminary Phytochemical Screening of the extract of Phyllanthus emblica and Adhatoda vasica Preliminary phytochemical analysis of the polyherbal extract containing Phyllanthus emblica and Adhatoda vasica revealed the presence of various bioactive compounds. A majority of biologically active phytochemicals were identified in the extracts, indicating that the medicinal plants are rich sources of secondary metabolites. These compounds are widely utilized in traditional medicine systems for the treatment and management of various ailments [12,13]. The presence of secondary metabolites such as polyphenols, flavonoids, tannins, terpenoids, steroids, glycosides, and phenolic compounds contributes to the antibacterial, anti-inflammatory, analgesic, and other therapeutic properties observed in these plants.

Table -2 Phytochemical studies of plant extract

+ Positive , - Negative

In the present investigation, nine bioactive components were identified from the acetone extract using Gas Chromatography-Mass Spectrometry (GC-MS). Among these, Squalene (12.05%) was found to be the most abundant compound, followed by dl-α-Tocopherol (11.69%) and the results were shown in figure 1. Previous studies have reported the phytochemical composition of the individual plant components. The ethanol leaf extract of Adhatoda vasica was found to contain terpene alcohols, alkaloids, vitamins, and sesquiterpene oxides, as identified through GC-MS analysis [14]. Similarly, GC-MS analysis of the ethanol leaf extract of Phyllanthus emblica revealed the presence of 22 components, with octadecanoic acid (22.93%) being the most prominent, followed by 9,12-octadecadienoic acid (14.99%) [15,16].

Figure 1: GC-MS Chromatogram of the extracts

The antibacterial activity of acetone extracts of Adhatoda vasica and Phyllanthus emblica at various concentrations of extracts using the stock as 1 mg/mL against the throat infection causing bacterial strains have been assessed in this study. Staphylococcus aureus and Klebsiella sp both show relatively strong inhibition, especially at higher concentrations (12 mm at 100µL).Klebsiella sp appears slightly more sensitive than Staphylococcus aureus at 60µL and 80µL.Pseudomonas sp shows poor sensitivity at lower concentrations (1 mm at 40µL) and moderate activity at higher levels (8 mm at 100µL), suggesting greater resistance compared to the other two strains.The tested sample (PAA) has effective antibacterial activity against gram-positive (S. aureus) and gram-negative (Klebsiella, Pseudomonas) bacteria. Greater efficacy is observed against S. aureus and Klebsiella sp, while Pseudomonas sp is more resistant, which is common due to its robust cell wall and efflux mechanisms [17]. The results were shown in table 3 and figure 2.

Table 3: Antibacterial activity of PAA Extracts against throat infection causing bacteria

Figure 2: A Klebsiella sps  B Pseudomonas sps

PAA exhibits significant antioxidant activity in a dose-dependent manner. Although it is less potent than ascorbic acid,used as a positive control it still shows considerable free radical scavenging potential, especially at higher concentrations. This suggests that PAA may be a useful natural antioxidant was shown in figure 3

Figure 3: Antioxidant Activity of PAA extracts