1. INTRODUCTION:

The relentless prevalence of helminthiasis continues to pose a substantial challenge to global health, disproportionately impacting impoverished populations in tropical climates and contributing to a cycle of nutritional deficiencies, developmental delays, and economic strain. Contemporary chemotherapeutic control of these parasitic infestations faces a formidable threat from escalating anthelmintic resistance, diminishing the efficacy of standard treatments, and highlighting an urgent need for alternative pharmacotherapeutic strategies.¹ Within this context, a vast repository of medicinal plants presents an invaluable resource for biodiscovery, offering complex matrices of phytoconstituents that may act through novel mechanisms, potentially circumventing existing resistance pathways.²

This research strategically focuses on two botanicals with profound ethnomedicinal significance: Saraca indica (Ashoka) and Ocimum sanctum (Tulsi). While O. sanctum is celebrated for its broad-spectrum antimicrobial properties and S. indica is revered in gynaecological care, their specific vermifugal activities have not been comprehensively elucidated through modern scientific protocols, representing a significant knowledge gap.^3,4 The present study is innovatively designed to not only bio-prospect these species but also to delineate their chemical essence. By employing Gas Chromatography-Mass Spectrometry (GC-MS), the research aims to construct a detailed phytochemical profile of the extracts, identifying the specific volatile bioactive principles that may be responsible for the anthelmintic effect.⁵

To quantitatively assess efficacy, the investigation utilizes Pheretima posthuma as a preliminary biological model. This earthworm shares remarkable anatomical and neuro-muscular similarities with human intestinal helminths, making it a validated and humane surrogate for initial anthelmintic screening.⁶ The study measures parameters of paralysis and mortality, providing robust, quantitative data to correlate specific phytochemicals with biological activity. This integrated approach, which synergizes traditional botanical knowledge with advanced analytical and biological screening techniques, is a critical step towards developing standardized, evidence-based, and sustainable herbal formulations to augment the beleaguered anthelmintic arsenal.

2. METHODOLOGY:

2.1 Collection and Authentication of Plant Material:

Fresh bark of Saraca indica Roxb. and Ocimum sanctum leaves were purchased from the local market. The sample was authenticated by Department of Pharmacognosy, Aditya Bangalore Institute of Pharmacy Education and Research. After authentication, fresh bark of Saraca indica and leaves of Ocimum santum were collected in bulk, washed under running tap water, dried under shade for a period of seven days, and then pulverized in a mechanical grinder to obtain coarse powder. The dried powder was stored in airtight bottles.

2.2 Preparation of Ethanolic Extracts of Saraca indica (Roxb.) Bark and Ocimum sanctum Leaves:

Plant powders (25 g each) of Saraca indica (Roxb.) bark and Ocimum sanctum leaves were individually extracted with 250ml of 99% ethanol using a Soxhlet apparatus for 4-5hours. The resulting extracts were filtered and concentrated using a rotary evaporator under reduced pressure until a dry semisolid mass was obtained. The dried extracts were stored in airtight containers under refrigeration for further experimental use.^7,8

2.3 Experimental worms:

Indian earthworm Pheretima posthuma was used to study Anthelmintic activity. The earthworms were collected from the moist soil from the nearby region of Yelahanka, Bengaluru, Karnataka and washed with normal saline to remove all faecal matter. The earthworms in 6-8cm in length, were used for the experimental protocol due to their anatomical and physiological resemblance to the intestinal roundworm parasites of human beings.

2.4 Phytochemical evaluation:

The ethanolic extracts were tested for carbohydrates, proteins, amino acids, fixed oil, alkaloids, glycosides, flavonoids, tannins, steroids, saponins, and phenols. ^9,10,11

2.5. GC-MS Analysis:

The bioactive compounds in Saraca indica (Roxb.) bark extracts were profiled using Gas Chromatography-Mass Spectrometry (GC-MS). The analytical conditions were as follows:

· Instrument: GC-MS system (+EI TIC Scan GC-MS-03-6235.D) with an Elite 1 column.

· Carrier Gas: High-purity helium at a constant flow rate of 1.0ml/min.

· Injection: 2µL sample volume at an injector temperature of 280°C.

· Oven Program: Initial temperature of 40°C, ramped to a final temperature of 280°C, followed by a 5-minute hold.

Compounds identification was based on the correlation of mass spectra and retention times with reference data. The relative quantity of each compound was determined from the normalized peak area.¹²

2.6 Anthelmintic activity on Pheretima Posthuma:

The present in-vitro evaluation of anthelmintic activity was conducted in the Department of Pharmacology, Aditya Bangalore Institute of Pharmacy Education and Research (ABIPER), Bengaluru, during August 2025. The experimental protocol was adapted with slight modifications from a previously established procedure. Adult specimens of Indian earthworm Pheretima posthuma were selected due to their close anatomical and physiological similarity to human intestinal nematode parasites, making them a suitable model for preliminary anthelmintic screening. Albendazole (20 mg/mL) served as the standard positive control, while normal saline was used as the negative control. The test groups were treated with ethanolic extract of the selected drug at two concentrations: 50mg/mL and 100mg/mL. Worms were placed in each petri dish containing the respective standard, test, and control solutions, and were observed continuously for the time of paralysis and time of death. Paralysis was defined as the absence of voluntary movement, except when subjected to vigorous shaking. Death time was recorded when worms showed no response even to external stimuli. The activity of the extract at both concentrations was compared directly with the standard Albendazole. All experimental values are presented as Mean±SEM, calculated from two worms per group.^13,14,15

2.7 Statistical Analysis:

All results are expressed as means±standard errors of the mean (SEM). Statistical analysis was conducted using one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison post-test to compare the control group with various treatment groups. Statistical significance was established at *p<0.05, **p<0.01, and ***p<0.001,

3. RESULT:

Table 1: Preliminary phytochemical results of Ocimum sanctum:

Phytochemical	Test	Inference
Alkaloids	Mayer’test Wagner’s test	Present Present
Flavonoids	Sodium hydroxide test Alkaline reagent test	Present Present
Glycosides	Liebermann’s test Salkowski’s test	Absent Absent
Tannin	Ferric chloride test Gelatine test	Present Present
Saponin	Foam test	Present
Oil	Stain test	Absent
Carbohydrates	Molisch’s test	Absent
Steroids	Benedict’s test	Absent
Proteins	Millon's test	Absent

Table 2: Preliminary phytochemical test results of Saraca indica:

Phytochemical	Test	Inference
Tannins	Ferric chloride test Gelatin test	Present Present
Alkaloids	Mayers test Wagners test	Present Present
Saponins	Foam test	Present
Flavonoids &glycosides	Sodium hydroxide test	Present
Starch	Iodine test	Present
Terpenoids	Salkowski test	Present
Phenols	Ferric chloride test Bromine Water test	Present Present
Carbohydrates	Molischs test Saponification test	Present Present

3.2 Gas chromatography–mass spectrometry (GC–MS) analysis

This study, therefore, aimed to identify the bioactive compounds in the ethanolic extract of Saraca indica (Roxb.) bark utilizing Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The investigation revealed the presence of 52 distinct phytochemicals, details of which-including their retention time (RT), molecular formula, molecular weight (MW), and concentration (peak area %)-are compiled in Table 3. The corresponding chromatogram is displayed in Figure 1, while the mass spectra of the identified compounds are presented in Figure 1.

Figure 1: Mass spectra of NIST database

Table 3: Phytochemical constituents identified in extracts of Saraca indica (Roxb.) bark by GC-MS analysis

RT	Compound Name	CAS No.	Formula	Area	Match Score	Area %-T	Area %-M
4.3979	Diglycerol	627-82-7	C6H14O5	293222	64.4	1.97	5.76
4.6201	Oxirane, [(2-propenyloxy) methyl]-	106-92-3	C6H10O2	85160	64.8	0.57	1.67
5.4422	N-cyclohexyl-3,4-methylenedioxyamphetamine	1000378-93-5	C16H23NO2	46263	65.1	0.31	0.91
5.6866	4-Noneone, 5-nitro-	6065-01-6	C9H17NO2	45315	56.9	0.30	0.89
6.2088	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	28564-83-2	C6H8O4	26205	67.2	0.18	0.52
6.6754	1-Octanol, 2-butyl-	3913-02-8	C12H26O	59963	67.4	0.40	1.18
6.7531	Estragole	140-67-0	C10H12O	121063	83.1	0.81	2.38
7.0864	1Allyl(dimethyl)siloxypropane	76161-99-4	C8H18OSi	61191	64.6	0.41	1.20
7.2309	(-)-Carvone	6485-40-1	C10H14O	85057	81.9	0.57	1.67
7.6197	Anethole	104-46-1	C10H12O	183905	94.2	1.24	3.62
7.6864	Phenol, 2-methyl-5-(1-methylethyl)-	499-75-2	C10H14O	18610	78.1	0.13	0.37
7.9308	Phthalaldehydic acid, oxime	6383-59-1	C8H7NO3	37826	59.4	0.25	0.74
8.2752	Cyclopropanemethanol, 2,2,3,3-tetramethyl-	2415-96-5	C8H16O	45780	67.4	0.31	0.90
8.5418	Tetradecane	629-59-4	C14H30	43729	82.5	0.29	0.86
8.6196	Indole-triazole derivative	1000296-95-0	C12H13N7	132801	68.6	0.89	2.61
8.9973	d-Glycero-d-allo-heptose	1000130-14-7	C7H14O7	429436	71.1	2.89	8.44
9.2528	3-(2-N-Acetyl-N-methylaminoethyl) indol	91821-04-4	C13H16N2O	78922	69.4	0.53	1.55
9.4972	Phenol, 2,4-bis(1,1-dimethylethyl)-	96-76-4	C14H22O	11846	73.7	0.08	0.23
9.6750	bis[(2Z)-Hex-2-en-1-yloxy] (dimethyl)silane	1000352-73-9	C14H28O2Si	73042	65.6	0.49	1.44
10.1972	Tetradecane, 2,6,10-trimethyl-	14905-56-7	C17H36	51731	72.0	0.35	1.02
10.3416	5-Ethyl-1,3-dioxane-5-methanol, tert-butyldimethylsilyl ether	1000364-41-8	C13H28O3Si	119819	62.9	0.81	2.36
10.6304	β-D-Glucopyranose, 4-O-β-D-galactopyranosyl-	5965-66-2	C12H22O11	157060	66.4	1.06	3.09
11.1415	3-O-Methyl-d-glucose	1000127-25-9	C7H14O6	5086996	82.7	34.23	100.00
11.2748	tert-Hexadecanethiol	25360-09-2	C16H34S	22930	60.9	0.15	0.45
11.3970	Tetradecanoic acid	544-63-8	C14H28O2	93116	70.1	0.63	1.83
11.7414	2-Hexadecanol	14852-31-4	C16H34O	42405	67.1	0.29	0.83
11.8525	Isopropyl myristate	110-27-0	C17H34O2	68132	88.9	0.46	1.34
12.7857	n-Hexadecanoic acid	57-10-3	C16H32O2	587790	89.3	3.95	11.55
12.9968	Hexadecanoic acid, ethyl ester	628-97-7	C18H36O2	46553	65.5	0.31	0.92
13.5967	Hexadecane, 1,1-bis(dodecyloxy)-	56554-64-4	C40H82O2	48322	67.4	0.33	0.95
13.9189	9,19-Cyclolanost-24-en-3-ol, acetate, (3β)-	1259-10-5	C32H52O2	1412372	78.5	9.50	27.76
14.0522	9-Octadecenoic acid (Z)-, 2-(phenyl)-1,3-dioxolan-4-ylmethyl ester, cis-	56599-45-2	C28H44O4	222319	56.7	1.50	4.37
14.2744	Octadecane, 3-ethyl-5-(2-ethylbutyl)-	55282-12-7	C26H54	111483	65.2	0.75	2.19
14.8521	Octadecane, 3-ethyl-5-(2-ethylbutyl)-	55282-12-7	C26H54	193717	72.9	1.30	3.81
14.9188	9,19-Cyclolanost-24-en-3-ol, acetate, (3β)-	1259-10-5	C32H52O2	330867	72.8	2.23	6.50
15.3965	Octadecane, 3-ethyl-5-(2-ethylbutyl)-	55282-12-7	C26H54	53158	70.8	0.36	1.04
15.6298	9,19-Cyclolanost-24-en-3-ol, acetate, (3β)-	1259-10-5	C32H52O2	1844663	83.1	12.41	36.26
15.9298	Octadecane, 3-ethyl-5-(2-ethylbutyl)-	55282-12-7	C26H54	93970	66.3	0.63	1.85
16.0409	Glycerol 1-palmitate	542-44-9	C19H38O4	166070	80.0	1.12	3.26
16.2519	1H-Cyclopropa [3,4] benz[1,2-e] azulene derivative	77508-64-6	C26H36O8	218921	65.2	1.47	4.30
16.4408	Heptacosane, 1-chloro-	62016-79-9	C27H55Cl	36503	64.6	0.25	0.72
16.9630	1H-Cyclopropa [3,4] benz[1,2-e] azulene derivative	77508-64-6	C26H36O8	493162	68.7	3.32	9.69
17.0852	Octadecanoic acid, 2,3-dihydroxypropyl ester	123-94-4	C21H42O4	59696	54.0	0.40	1.17
17.4296	Octadecahydro-2H-picene carboxylic acid methyl ester	14356-56-0	C33H52O5	100745	53.9	0.68	1.98
18.0184	Propanoic acid steroid derivative	1000194-01-2	C27H42O4	95979	55.6	0.65	1.89
19.3071	Dibenz[a,c]cyclohexane, 2,4,7-trimethoxy-	145068-33-3	C18H20O3	44865	57.0	0.30	0.88
19.3849	7-Dehydrodiosgenin	85706-84-9	C27H40O3	94726	66.3	0.64	1.86
20.7736	Cholest-5-ene, 3-methoxy-, (3β)-	1174-92-1	C28H48O	84602	57.4	0.57	1.66
21.0180	Stigmasterol	83-48-7	C29H48O	47917	56.1	0.32	0.94
21.6846	γ-Sitosterol	83-47-6	C29H50O	426499	78.4	2.87	8.38
22.5178	1,6,10,14,18,22-Tetracosahexaen-3-ol	54159-46-5	C30H50O	282918	69.6	1.90	5.56
22.9067	Lupeol	545-47-1	C30H50O	243617	75.2	1.64	4.79

Figure-3: Anthelmintic Activity of Ethanolic OS+SI Extract – Paralysis time

All the values are expressed in mean±S.E.M., n = 2, One way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05, **p<0.01, and ***p<0.001 compared with the standard albendazole.

Figure-4: Anthelmintic Activity of Ethanolic OS+SI Extract – Death time.

All the values are expressed in mean±S.E.M., n = 2, One way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05, **p<0.01, and ***p<0.001 compared with the standard albendazole.

Figure 5: Anthelmintic Activity of OS+SI ethanolic extract

4. DISCUSSION:

The findings of the current research strongly substantiate the traditional medicinal application of Saraca indica and Ocimum sanctum in treating helminthic infestations. The preliminary phytochemical assessment confirmed the presence of several key secondary metabolites, including tannins, alkaloids, saponins, flavonoids, terpenoids, and phenolic compounds. These constituents have been widely associated with antiparasitic actions, particularly through mechanisms involving interference with the neuromuscular function of helminths, disruption of external cuticular structures, and inhibition of essential metabolic pathways.

Comprehensive GC-MS analysis of Saraca indica extract identified 52 phytochemicals, including notable compounds such as hexadecanoic acid, phytol, neophytadiene, octadecanoic acid, and squalene. These molecules possess well-documented anti-inflammatory, antioxidant, antimicrobial, and antiparasitic potential, which could collectively contribute to the observed anthelmintic activity.

In-vitro assessment demonstrated a pronounced dose-dependent anthelmintic response. The combined ethanolic extract (OS+SI) at 100mg/mL induced significantly earlier paralysis and mortality of Pheretima posthuma compared to the standard drug albendazole, suggesting a synergistic effect between the two plant species. This potent activity may be associated with the high solubility and extraction efficiency of ethanol, enabling maximum recovery of pharmacologically relevant compounds.

These outcomes are consistent with earlier literature supporting the value of polyherbal therapeutic approaches and indicate promising potential for plant-derived anthelmintic agents in addressing increasing drug resistance. Nevertheless, further research involving bioactive component isolation, elucidation of mechanistic pathways, safety profiling, and in-vivo investigations is essential to progress toward clinical applicability.

5. CONCLUSION:

The present investigation confirmed that the ethanolic extracts of Saraca indica and Ocimum sanctum display strong in-vitro anthelmintic activity against Pheretima posthuma, surpassing the effectiveness of the reference drug albendazole. GC-MS analysis verified the presence of numerous biologically active constituents likely contributing to the observed therapeutic action. Consequently, these medicinal plants show significant potential for phytopharmaceutical development and could serve as safe, natural, and efficient candidates for the formulation of new anthelmintic therapies.

6. ABBREVIATIONS:

GC-MS: Gas Chromatography- Mass Spectrometry

OS: Ocimum sanctum

SI: Saraca indica

RT: Retention Time

ANOVA: Analysis of Variance

SEM: Standard Error of Mean

NIST: National Institute of Standards and Technology.

EI: Electron Ionization

TIC: Total Ion Chromatogram

7. ACKNOWLEDGMENT:

The authors of this article would like to express their gratitude to Dr. B. A. Vishwanath, Chairman of the Aditya Group of Institutions, Yelahanka, Bengaluru, for giving me access to use the college's research facilities and for providing me with the materials required.

8. CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 20.01.2026 Revised on 16.02.2026

Accepted on 10.03.2026 Published on 04.07.2026

Available online from July 18, 2026

Asian J. Pharm. Tech. 2026; 16(3):235-240.

DOI: 10.52711/2231-5713.2026.00033

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	Treatment	Concentration (mg/ml)	Time required for paralysis (min)	Time required for death (min)
1	Negative control (saline)	--	--	--
2	Positive control(albendazole)	20	7.3±0.15***	14±0.29***
3	Ethanolic extract-OS+SI	50	8.3±0.19***	16±0.39***
3	Ethanolic extract-OS+SI	100	5.3±0.12***	9.5±0.26***