1. INTRODUCTION:

1.1 Background: Helminth infections are a major global health issue, especially in tropical and subtropical areas. Helminth infections, caused by parasitic worms such as roundworms, hookworms, tapeworms, and flukes¹. These parasites can cause symptoms ranging from mild discomfort to severe illness². These infections affect millions of people worldwide, leading to significant morbidity, malnutrition, and diminished quality of life.

Standard treatment involves anthelmintic medications, which expel these parasites by either killing them or inhibiting their growth³. Common synthetic anthelmintics, such as Albendazole and Mebendazole have proven effective but these medications can cause side effects like abdominal pain, nausea, and dizziness, drug resistance, and limited accessibility in low-resource settings underscore the need for alternative treatment options such as polyherbal formulations, which may offer effective and safer options for managing helminth infections^4,5.

Polyherbal formulations, which combine multiple plant extracts, offer a promising alternative to conventional anthelmintic drugs. These formulations leverage the synergistic effects of various plant compounds, potentially enhancing efficacy and reducing side effects. Syrups, in particular, present an advantageous dosage form for populations that have difficulty swallowing tablets or capsules, such as children and elderly individuals. Despite the traditional use of herbs in treating helminth infections, there is limited scientific investigation into the effectiveness of polyherbal syrups specifically for this purpose. Exploring these formulations could provide new insights and viable alternatives to current treatments^6,7. For the preparation of polyherbal syrup formulations, the hydroalcoholic leaves extract of Trigonella foenum-graecum (Common name: fenugreek), Coleus amboinicus (Common name: ajwain) and Murraya koenigii (Common name: Curry leaves) were used.

1.2 Objective:

The primary objective of this study is to evaluate the anthelmintic activity of polyherbal syrup formulations through comprehensive in vitro assessments. This research aims to formulate various polyherbal syrups, conduct thorough phytochemical screening, and compare their efficacy with standard treatments such as Albendazole. By investigating these alternative formulations, this study seeks to contribute to the development of more accessible and effective treatment options for helminthiasis, potentially offering improved health outcomes for affected populations.

2. MATERIALS AND METHODS:

2.1 Plant Material:

Three plants were selected for this study. The selected plants included Trigonella foenum-graecum, Coleus amboinicus, and Murraya koenigii. The plants were chosen based on their traditional use in treating parasitic infections and their content of key phytochemicals such as phenolic compounds, tannins, and flavonoids. The leaves of Trigonella foenum-graecum and Murraya koenigii were collected from the APMC market, Sector-19, Vashi, Navi Mumbai and the leaves of Coleus amboinicus were collected from Hruturaj Green Nursery, Plot 125, Sector-26, Seawoods, Navi Mumbai Maharashtra. The leaves of all plants were authenticated by Dr. Rajendra D. Shinde, Principal and Head of the Department of Botany and Director of the Blatter Herbarium, St. Xavier's College, Mumbai, India (400001). He provided specimen no. (Shah- 1600 of G. L. Shah.), (MRA-1582 of M.R. Almeida) and (R-3594 of R.R. Fernandez) for reference. The plant materials were dried, ground into a fine powder, and stored in airtight containers until use.

2.2 Preparation of Hydroalcoholic Leaves Extract:

For the preparation of hydroalcoholic extract, about 70 grams of dried coarsely powdered leaves of each plant was filled in the thimble chamber of the Soxhlet apparatus and extraction process was carried out by using 500 ml of 70% ethanol and 30% water (V/V) mixture for three days. The solubility of both solvents is good for the majority of organic compounds. The solvents were heated in the round bottom flask, vaporized into the sample thimble, condensed in the condenser, and then dripped back into the flask. The procedure was resumed once the liquid content reached the syphon arm and drained back into the bottom flask. This process was repeated twice to obtain required quantity of extract. After being concentrated by evaporation at 70°C for 8 hr, the extract was dried. At last, % yield of the extract was calculated and kept it at room temperature prior to phytochemical screening^8.9.

2.3 Phytochemical Screening:

Phytochemical screening was conducted to identify the presence of phenolic compounds, tannins, flavonoids, and other relevant phytochemicals in the plant extracts. Standard qualitative tests were performed:

Phenolic Compounds and Tannins: Ferric chloride test, Gelatin test, Alkaline reagent test, and Bromine water test.

Flavonoids: Lead acetate test, Shinoda test and Magnesium turning test and Zinc hydrochloric acid reduction test.

The results were recorded based on color changes and precipitate formation, and compared to known standards^10,11.

2.4 UV Spectrophotometry:

UV spectrophotometry was employed to identify bioactive components in the hydroalcoholic leaves extract of three plants. Samples were prepared by diluting the extracts with water and ethanol. System calibration was automatically programmed in the Spectrophotometer. LabSolutions UV-Vis software provided by Shimadzu is used for analysis and interpretation of spectral data. The UV spectra were recorded in the range of 200-400 nm using a UV-Vis spectrophotometer (Shimadzu UV-1900 i). The characteristic absorption peaks corresponding to bioactive compounds were analyzed^12,13.

2.5 Preparation of Syrup:

Two formulations of polyherbal syrup: Formulation 1 (F-1) and Formulation 2 (F-2) were prepared from simple syrup using a standardized method.

2.5.1 Preparation of Simple Syrup:

Simple syrup was prepared by following the process as given in IP. 66.7 gm of sucrose was weighed and added to purified water and heated until it dissolved with occasional stirring. Sufficient boiling water was added to produce 100ml¹⁴.

2.5.2 Preparation of Polyherbal Syrup:

Simple syrup was used a base for the preparation of the polyherbal syrup formulations according to the formula given in Table-1.

Table 1: Formulations of Polyherbal Syrup

Ingredients	Function	Formulation
Ingredients	Function	F₁	F₂
Trigonella foenum-graecum,	Active Drug	500mg	1000mg
Coleus amboinicus	Active Drug	500mg	100 mg
Murraya koenigii	Active Drug	500mg	1000 mg
Propylene Glycol	Solubilizer	6.0ml	6.0 ml
Methyl Paraben	Preservative	0.025gm	0.025 gm
Propyl Paraben	Preservative	0.0025gm	0.0025 gm
Glycerine	Thickening agent	0.5 ml	0.5 ml
Simple Syrup	As a Base	Up to 100 ml	Up to 100 ml

Method:

Appropriately weighed quantity of each ingredient was taken. The extract was thoroughly combined with a small amount of water in a mortar and pestle. Required quantity of preservatives (methyl paraben and propyl paraben) and solubilizer (propylene glycol) was mixed with the extract. Simple syrup was added to the content of mortar and mixed together. Glycerine was added as a thickening agent and then finally make up the volume upto 100 ml with distilled water. A homogenous mixture was obtained. Solubility was checked by observing the clarity of solution visually. Syrup was transferred into a bottle, labelled appropriately and stored until further use¹⁵.

2.6 Evaluation of Polyherbal Syrup Formulations:

2.6.1 Organoleptic Properties:

The colour, taste, appearance and odour of the polyherbal syrup were immediately determined using sensory and visual examination.

2.6.2 pH Determination:

The pH of polyherbal syrup was determined by using digital pen-style pH meter and pH paper¹⁶.

2.6.3 Determination of Density of Syrup:

The empty bottle of known volume was weighed (W₁) and then filled with syrup and weighed again (W₂). The difference between the weight of the empty bottle and the bottle containing the syrup was recorded (W₃) and density was calculated using the given formula:

Weight of Syrup

Density of Syrup = ------------------------------------

Volume of Bottle

2.6.4 Determination of Specific Gravity of Syrup:

The pycnometer was used to determine specific gravity (g/mL) of the syrup formulations. The weight of the empty pycnometer and its weight when filled with water were recorded as W1 and W2 respectively. The weight of the syrup in the pycnometer was recorded as W3. Mass of syrup was calculated by subtracting the weight of the empty pycnometer from the weight of the syrup in the pycnometer. Mass of water was calculated by subtracting the weight of the empty pycnometer from the weight of pycnometer filled with distilled water. Specific gravity was computed using the formula below;

Mass of Syrup

Specific Gravity of Syrup = --------------------------------

Mass Of Distilled Water

2.6.5 Viscosity:

Using an Ostwald viscometer, the viscosity of polyherbal syrup formulations was measured. The viscometer was mounted vertical position on a suitable stand. Syrup was filled in to the viscometer up to mark A. The time was counted for syrup to flow from A to mark B. Viscosity was measured in triplicate.

2.6.6 Determination of Crystal Growth:

The crystal growth was determined after 24 hours.

2.6.7 After Taste:

The taste is strong and remains unchanged over the week except for the ambient temperature sample. Healthy male and female candidates, were selected to participate in the evaluation of taste. About 1 ml of polyherbal syrup formulations was given to selected candidates and ask for the taste.

2.6.8 Stability Studies:

The aim of stability studies was to guarantee that the polyherbal syrup formulations can be used and can continue to have the same properties over time. The prepared polyherbal syrup formulations were stored at room temperature, in the refrigerator and at accelerated temperature (40°C) for 30 days and assessed visually for presence/absence of growth or crystals.

2.7 In vitro Anthelmintic Assays:

To conduct in-vitro anthelmintic activity, an Indian adult earthworm experimental model was selected. Due to the anatomical and physiological resemblance with the intestinal round worm parasite of humans Pheretima prostuma earthworms (6-10 cm long and 1-2 cm wide) were used as animal model for the screening^17,18,19.

2.7.1 Collection of Pheretima prostuma Earthworms: Pheretima prostuma earthworms were collected from Vasudha Vermicompost by Begreenss Raigad, Navi Mumbai, Maharashtra.

Figure 1: Pheretima prostuma

2.7.2 Screening of Anthelmintic Activity:

The earth worms were thoroughly washed with normal saline (0.9% w/v of NaCl). All the test solutions and standard drug solutions were prepared freshly before starting the experiment.

Experimental Design for Anthelmintic Activity:

The earthworms were divided into seven groups containing 6 earthworms in each group. The earthworms were kept in glass petridishes for the respective treatments.

· Group 1: served as normal control and received 10 ml normal saline solution.

· Group 2: served as standard group and received 10 ml of Albendazole Oral Suspension.

· Group 3: served as test group 1 and received 10ml of polyherbal syrup formulation (F-1).

· Group 4: served as test group 2 and received 10ml of polyherbal syrup formulation (F-2).

· Group-5: served as test group 3 and received 10ml of hydroalcoholic extract of Trigonella foenum-graecum leaves (400mg/ml).

· Group-6: served as test group 4 and received 10ml of hydroalcoholic extract of Coleus amboinicus leaves (400mg/ml).

· Group-7: served as test group 5 and received 10ml of hydroalcoholic extract of Murraya koenigii leaves (400mg/ml).

Observations were made for the time taken for paralysis (immobility) and complete death of the earthworms to be induced by every treatment. Death time was recorded when the worms lost their mobility followed with their body color fading away. The readings were verified by repeating the experiment.

2.8 Statistical Analysis:

The efficacy of the polyherbal syrups was compared to that of Albendazole using statistical methods. All Statistical calculations were performed using Microsoft excel. The data are expressed as Mean±SEM. Statistical significance of the differences between the groups was analyzed using Student’s t-test. P values were calculated in each case and accordingly interpretation was carried out. Statistical analysis of both paralysis time and death time of the earthworms in case of standard and Formulation 1 and 2 of polyherbal syrup were done. The comparative analysis aimed to determine whether the polyherbal syrups offer comparable or superior anthelmintic activity.

3. RESULTS:

3.1 % yield of Hydroalcoholic Leaves Extracts of Herbal Plants:

The % yields of the hydroalcoholic leaves extracts of different plants are presented in table 2. The % yield of hydroethanolic leaves extract of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii was calculated by using following formula:

Weight of dry extract

% Yield = -----------------------------------------------X 100

Weight of dry plant biomass

Table 2: % Yield of Hydroalcoholic Leaves Extracts of Different Plants

S. No.	Plant	Weight of Dry Plant taken (gm)	Weight of Dry Extract obtained (gm)	Yield (%)
1	Trigonella foenum-graecum	140	15.39	10.99
2	Coleus amboinicus	100	18.5	18.5
3	Murraya koenigii	140	24.26	17.32

Figure 2: Hydroalcoholic Leaves Extract of Plants

3.2 Phytochemical Screening:

The preliminary phytochemical screening of hydroalcoholic leaves extract of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii revealed the presence of phenolic compounds, tannins, flavonoids, alkaloids, phytosterols and terpenoids.

3.3 UV Spectrophotometry:

UV spectrophotometric analysis identified several bioactive components in the hydroalcoholic leaves extracts of plants. Characteristic absorption peaks corresponding to known anthelmintic compounds were observed:

Trigonella foenum-graecum showed the absorption peaks at 279, 283, 293, 299, 312, 357nm with the absorption 0.966, 1.012, 0.766, 0.754, 0.454, 0.433 respectively. Coleus amboinicus showed the absorption peaks at 280, 293, 296, 380, 390 nm with the absorption 0.596, 0.600, 0.267, 0.487, 1.043 respectively. Murraya koenigii showed the peaks at 284, 290, 302, 381, 397nm with the absorption 1.076, 0.667, 0.273, 0.477, 1.063 respectively. According to the previous studies, these absorption bands are characteristic for phytochemicals. An investigation conducted by Patrica et al., (2013) explained that flavonoids and phenolic compounds fraction showed maximum absorption in the range of approximately 300-380nm and 290-350nm in UV spectroscopy which is in resemblance to our findings. These findings confirm the presence of key bioactive compounds contributing to the anthelmintic activity.

3.4 Preparation of Polyherbal Syrup Formulations (F-1 and F-2):

Two formulations of polyherbal syrup: Formulation 1 (F-1) and Formulation 2 (F-2) were prepared (Figure 3).

Figure 3: Polyherbal Syrup Formulations

3.5 Evaluation of Polyherbal Syrup Formulations:

Both prepared formulations (F-1 and F-2) of polyherbal syrup were subjected for evaluation parameters. The results of evaluation parameters are presented in table 3.

Table 3: Evaluation Parameters of Both Polyherbal Syrup Formulations

S. No.	Parameters	F-1	F-2
1	Color	Dark Brown	Dark Brown
2	Odour	Aromatic	Aromatic
3	Taste	Mildely sweet	Mildely sweet
4	Appearance	Clear	Clear
5	pH (By Digital pen style pH meter)	6.6	6.4
6	pH (By pH Paper)	6.7	6.7
7	Density	1.77	1.63
8	Specific Gravity	1.240	1.253
9	Viscosity	16.75 cps	13.10 cps
10	Crystal Growth	No Growth	No Growth
11	After Taste	Same	Same
Stability Studies
12	Change in color	No Change	No Change
	Odour	No Change	No Change
	Physical Separation	No Change	No Change

3.6 Results of In Vitro Anthelmintic Activity:

Both formulations F1 and F2 of polyherbal syrup exhibited a significant paralysis effect and complete death of Pheretima prostuma earthworms. F-2 formulation of polyherbal syrup was more effective than the standard Albendazole suspension as it showed paralysis and death of earthworm in less time while the individual herbal extracts of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii leaves were found not to be as effective as the standard.

Table 4: Anthelmintic activity of Polyherbal Syrup Formulations and Hydroalcoholic Leaves Extracts of Different Herbs

S. No.	Treatment	Concentration (mg/ml)	Paralysis Time (min/sec)	Death Time (min/sec)
1	Control (Normal Saline)	10 ml (0.9% NaCl Solution)	No Paralysis	No Death
2	Standard (Albendazole Oral Suspension IP)	10 ml (200 mg/5ml)	5.32± 0.45	18.86± 2.15
3	F1- Formulation	10 ml (400 mg)	5.18± 0.23	17.62± 2.06
4	F2- Formulation	10 ml (400 mg)	5.02± 0.24	16.65± 1.63
5	Trigonella foenum-graecum Hydroethanolic Extract	400 mg	7.73± 0.75	25.46± 0.67
6	Coleus amboinicus Hydroethanolic Extract	400 mg	10.86± 0.41	26.15± 0.49
7	Murraya koenigii Hydroethanolic Extract	400 mg	11.90± 0.19	22.83± 0.94

Note: All values represent Mean ± SEM: n=6 in each group, Comparisons made between standard versus treated groups.

Figure 4: Anthelmintic Activity of Polyherbal Syrup Formulations

Figure 5: Graphical Presentation of Anthelmintic Activity of Polyherbal Syrup Formulations

Statistical analysis of the anthelmintic activity showed that both polyherbal syrups had efficacy comparable to that of Albendazole. Statistical analysis of the anthelmintic activity showed that both polyherbal syrups had efficacy comparable to or exceeding that of Albendazole.

Significant Differences:

T-test results revealed significant differences between the polyherbal syrups and Albendazole (p < 0.05).

Effectiveness:

F-2 formulation of polyherbal syrup demonstrated higher effectiveness in terms of both paralysis and mortality rates compared to Albendazole.

4. DISCUSSION:

The results of this research work demonstrate that polyherbal syrups exhibit significant anthelmintic activity, with both formulations showing efficacy comparable to or exceeding that of Albendazole. F-2 formulation of polyherbal syrup was more effective than the standard Albendazole suspension as it shows paralysis and death of earthworm in lesser time while the individual herbal extracts of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii leaves were found not to be as effective as the F-2 formulation and standard Albendazole. The observed anthelmintic effects, including paralysis and mortality of the helminths, suggest that these polyherbal syrups could be effective alternatives to conventional treatments. The positive outcomes indicate that combining multiple plant extracts in syrup formulations can enhance the therapeutic potential, likely due to the synergistic effects of various bioactive compounds. Phytochemical screening revealed the presence of key compounds such as phenolics, tannins, and flavonoids in the plant extracts used for syrup formulation^20,21. These compounds are known for their biological activities. Phenolic compounds and flavonoids, in particular, have been shown to exhibit anthelmintic properties through mechanisms such as inhibition of enzyme activity and interference with parasite metabolism^22,23. The UV spectrophotometric analysis confirmed the presence of specific bioactive compounds, further supporting their role in the observed anthelmintic activity. The correlation between the concentration of these compounds and the efficacy of the syrups highlights their importance in the formulation’s effectiveness. When compared to Albendazole, the polyherbal syrups performed competitively. While Albendazole remains a widely used and effective anthelmintic drug, the polyherbal syrups provided a promising alternative with comparable or even superior efficacy in some cases. The statistical analysis indicated significant differences between the both formulations of polyherbal syrup and Albendazole, with F-2 formulation achieving higher effectiveness. This suggests that polyherbal syrups could offer a viable complementary or alternative treatment option, potentially benefiting from the diverse action mechanisms of the multiple plant extracts used. The current research investigation provides a golden opportunity for the budding herbal researchers to explore the formulation for its Phytochemistry and its mechanism of action.

5. CONCLUSION:

This research is designed to explore the potential of polyherbal syrup formulations as effective, safe, and affordable alternatives for the treatment of helminthic infections. By focusing on scientifically validating these formulations, the study aims to contribute to the growing body of evidence supporting the use of herbal medicine in modern healthcare. The formulated polyherbal syrup formulations can significantly work in long way to help people to prevent helminthic infections. Polyherbal syrup formulations combine multiple herbs, each with distinct active compounds that can work synergistically. This synergy can enhance the overall therapeutic efficacy compared to single-herb treatments. Furthermore, we can assure that no harmful ingredients are present in polyherbal syrup formulations. The polyherbal syrup is better than albendazole suspension as it shows better results. The phytochemical analysis revealed that key compounds such as phenolics, tannins, and flavonoids contribute to the anthelmintic activity. It has been medically demonstrated that the natural herbs utilized in the current formulations may prevent the other infection also. The findings of this study suggest that polyherbal syrups could serve as viable alternatives to conventional anthelmintic treatments, particularly in settings where traditional medications are less accessible. These syrups, being easier to administer, may be especially beneficial for pediatric and geriatric populations. The present study is crucial for developing an affordable, efficient polyherbal syrup for low socioeconomic communities. However, since this study was brief, longer studies with larger sample sizes are necessary.

6. ACKNOWLEDGEMENT:

Authors are thankful to NCRD Sterling Institute of Pharmacy for providing the resources and support necessary for the completion of this research.

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Received on 25.09.2024 Revised on 21.01.2025

Accepted on 26.03.2025 Published on 10.02.2026

Available online from February 16, 2026

Research J. Pharmacy and Technology. 2026;19(2):572-578.

DOI: 10.52711/0974-360X.2026.00084

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