1. INTRODUCTION:

Genus Terminalia has a massive number of the tree of flowering plants belonging to Combretaceae, distributed with more than 200 different species throughout the globe. The name of the genus 'Terminalia' is derived from the Latin word "Terminus," citing that leaves present at the correct tip of shoots. The plants from this genus are essential due to different phytoconstituents such as tannins, terpenoids, flavonoids, and aromatic principles¹. This genus different species such as arjuna², chebula³ having huge medicinal utility.

Its efficacy cannot be avoided in famous formulation triphala churna⁴. The focused aim of this investigation is to access the helminthic activity of T. coriacea nanoemulsions against Haemonchus contortus (H. contortus) and Pheretima posthuma (P. posthuma) on egg hatch, larval motility, and adult worm motility test.

These are antimicrobial, hepatoprotective ⁵ ⁶ anti-epileptic activity⁷, wound healing ⁸, anti-nociceptive ⁹, anticancer and antibacterial ¹⁰ evidence. T. coriacea plants from the Terminalia genus native to India have various important traditional uses in treating ulcers, wound healing, cardiac stimulants, and diarrhoea⁶, and hepatoprotective ¹¹. It grows in an embossed part of a dry, moist forest in south India at 1000 m. The geographical origin is south and east in India, Nepal, Thailand, Bangladesh, Laos, Vietnam, Myanmar, and Cambodia. The helminth parasites in sheep of the Medak district are H. contortus and P. posthuma ¹².

2. MATERIALS AND METHODS:

2.1 Raw materials collections:

Bark collection was done from the tree of Tirumala Hills situated in Chittoor district (AP), and Dr. K. Madhava Chetty authenticated the plant materials. An authentication letter (Voucher specimen No.-1109) is submitted at Gitam School of Pharmacy, Rudraram (V), Patancheru (M), Sangareddy (D) Hyderabad (TS).

2.2 Extraction of Plant Materials:

The bark pieces were exposed to shade drying for a week in the lab to remove excess moisture; after drying, they were cut into small sizes and made powder subjected to extraction by Soxhlet apparatus¹³. The 100 gm. of coarse powder were extracted over 72 hours with successive solvents of variable polarity, including chloroform, petroleum ether, ethanol, and distilled water. After the extraction, the liquid was filtered and dried, and the concentrated extract was by air ¹⁴ ¹⁵.

2.3 Preparation of the nanoemulsion containing the T. coriacea Bark ethanolic extracts:

The formulation of Nanoemulsion having T. coriacea bark extracted by ethanol involves high energy technique of emulsification. Isopropyl myristate (oil), phenyl hydroxyethyl cellulose ether and sorbitan oleate (surfactant). The oil phase, which contained 0.13 % T. coriacea solubilized in propylene glycol (2%), isopropyl myristate 20 %, 13% phenyl hydroxyethyl cellulose ether and sorbitan oleate (7.2:1.5), and preservative phenonip 0.7%, was heated to 80 degrees Celsius. At 600 rpm and 80°C, the water phase (65.95% water) decreased during stirring. The heating was turned off after five minutes, and the mixture was agitated for three minutes. The nanoemulsion was then mixed to Sepigel (0.8%) and put in an aluminum-coated tube after cooling. ¹⁶

Figure 1: A schematic illustration of the Terminalia coriacea (TC) ethanolic bark extract nanoemulsion formulation procedure. To create a stable nanoemulsion with the ideal particle size, the bark is extracted in ethanol, combined with water, surfactants, and an oil phase (isopropyl myristate), and then stirred rapidly and cooled, and subjected for particle size analysis and zeta potential.

2.4 Characterization of nanoemulsion:

Physical stability and visual aspects After being centrifuged for 30 minutes at 2,500 g, the nanoemulsion examined for viewable characteristics such colour, creaming, phase separation and stability¹⁷.

2.5 Particle size investigation:

The determination of size for droplets of nanoemulsion were conducted. This was performed by employing photon correlation spectroscopy at 25°C. The samples diluted by ultrapure water at a ratio of 1:100, prior to investigation. Every data point in the study reflects the average value of three distinct observations made at a consistent 173° angle using a laser 633nm¹⁸.

2.6 Zeta potential analysis:

Malvern Instruments' Zetasizer NanoZS ZEN 3600 was used to test zeta potential. Prior to being analysed in triplicate, all samples were dissolved with water (ultrapure) in 1:100 ratio ¹⁹.

2.7 Morphological evaluation:

The JEM-1200 EXII apparatus, manufactured by JEOL in Tokyo, Japan, was employed to investigate the morphological features of the nanoemulsion using the technique of transmission electron microscopy (TEM). The samples diluted using distilled water at 1:100 ratio. A 400-mesh copper grid, coated with carbon and covered with formvar/carbon, was delicately treated with a small quantity of the mixture. Later on, the grid underwent treatment with a 2.0% uranyl acetate solution in water for a duration of 10 minutes. The investigation was carried out at room temperature using transmission electron microscopy (TEM) with voltage of 80 kilovolts (kV) ²⁰.

2.8 Rheological behaviour analysis:

For this investigation a viscometer used and investigated at 25°C. There are three steps involved inhibiting shear rate, increasing and maintaining of it for every 180 seconds. The software Rheowin utilized to examine the thixotropic behaviour²¹.

2.9 Investigations on the nanoemulsion for in vitro release:

The markers from formulated Nanoemulsion of T. coriacea extract were screened by diffusion cells franz type. Dialysis membranes (Sigma-Aldrich Co.) were employed for the release investigations. In order to establish sink conditions, the receptor media was a 70% hydroalcoholic solution. The donor chamber was occupied by the formulations that were undergoing testing. Then, 1 mL samples were removed from the compartment of receptor and substituted with the similar volume of receptor medium at 1, 2, 4, 6, 8, 12, and 24 hours into the analysis²².

The processes employed to filter all samples prior to HPLC analysis. The total quantity of markers released by the membrane per diffusion region was depicted as a role of time. The release equation were employed to suit the release profiles generated by the formulations ²³.

2.10 In vitro tests:

Eggs were screened out from the waste products (feces) of sheep which are already infected. The same was used for infective larvae culture to perform a larval motility test (LMT). Gastrointestinal tract (GIT) nematode presence was verified by eggs counting (Egg each gram feces EPG)²⁴.

2.10.1 Egg hatch test:

The egg hatch investigation was done ²⁵described with slight changes. Concisely, the infected sheep were identified from the Sangareddy district village area, and 20 gm of feces were taken. It was washed in running water to remove earthy matter. Different sieves (150-, 90-, and 20-mm size) were used to screen the eggs. In tiny pores, size sieves had eggs because of their size. These eggs were retrieved by floatation with supersaturated saline. The retrieved eggs cleaned three to four rounds using purified water and centrifuged for 5 minutes to get egg suspension. Microdilution plates were used to distribute suspension (100 µL) that contained near about 100 embryos. The TcNano was taken to the plates with different dilutions of 50 %, 25%, 12.5%, 6.25%, and 3.25% (v/v). The standard drug Albendazole (2 mg/mL) was given as control group and for negative control group only distilled water provided. The investigations were conducted for 24 hours at a temperature of 27°C. The egg numbers and initial larvae were tallied as per Powers et al.²⁶. The percentage decline of egg hatching was expressed in the results ²⁷ ²⁸ ²⁹.

2.10.2 Larval motility test:

The movement of third-stage larvae with or without plant extract needs evaluation. The eggs are cultured as per Ueno and Goncalves³⁰ with slight changes. Concisely, 20 gm of infected feces with eggs at approximately 2000 EPG was homogenized. The moisture was maintained by adding distilled water. The larvae of third-stage (L3) were accumulated by warm water migration from 1-week incubated culture at room temperature³¹ ³².

Afterward, 50 µL suspension was subjected to microdilution plates, and TcNano was mixed as the egg hatch test method. The setup was kept for incubation at 27°C up to 24 hrs. Once incubated, the motile and non-motile larvae were identified and counted. Results revealed a % inhibition of larval movement³³.

2.10.3 Test for the motility of adult worms:

The Indian healthy adult earthworm P. posthuma was selected for the study³⁴ ³⁵. The worms were in uniform size during the experiment. They were collected from the Nizam Medical College, Hyderabad. 2.5 gm of desiccated extracts were dissolved in 25 mL in normal saline (with 1% gum acacia) to prepare samples for anthelmintic activity. Different concentration of solution was prepared to achieve a dose range of low dose 10 mg/mL, median dose 50, and high dose 100 mg/mL. The uniform size of the earthworm was selected and kept in six different groups for Standard, pet ether, chloroform, ethanolic, and hydro alcoholic extracts. The observations were focused on the time taken by worm to paralyzed or die. It was claimed that paralysis happens when the worm’s movement not noticed even in normal saline. The worms' demise was signalled by their loss of movement, and dimming colour of their body³⁶.

2.11 LCMS Analysis of Ethanolic extract of T. coriacea Bark:

The ethanolic bark extract underwent LCMS analysis using the LC MS Qtrap Instrument with ExionLC AD UHPLC System at, Visakhapatnam. The LCMS analysis was done with the calibrated extract where the efficient effects was observed. The ethanolic extract of bark was subjected for the analysis. Before the GCMS analysis was also carried out by the author for the same plant materials for the detection of volatile content. ³⁷.

The specific constituents in the fraction of ethyl acetate are revealed by the LC-MS investigation of the methanol fraction. Nevertheless, Astilbin was detected in the methanol fraction to have a variety of biological functions, including antioxidant, antifungal, anticarcinogenic, and anticonvulsant properties³⁸.

The metabolites contained in the extract were analyzed using reverse phase LC-MS. MicrOTOF-Q III was used to conduct the mass spectrometry. Untargeted analysis was employed to analyze the phytoconstituents present in O. indicum³⁹.

The bioactive compounds in the N. cadamba leaf were identified through a liquid chromatographic analysis. The identification of 32 phytoconstituents reported⁴⁰.

The chemical compound from P. anserina extracts screened by using LC-MS. This plant revealed various important phytoconstituents⁴¹. The Ephedra species also shown 11 phenolic components by these techniques⁴².

Table 1: Constituents detected from LC-MS of ethanolic bark extract of TC.

1	Glyceraldehyde	7	Norbuprenorphine-d3
2	Phenylcoumarin	8	Quinazolinamine
3	Cucurbitacin E	9	Triflupromazine
4	Surfactant compound	10	Flavanone
5	Mephenytoin	11	Methyl coumarin acetate
6	Serpentine mineral cation

2.12 Statistical analysis:

The data given as the mean and standard error mean was statistically compared by multiple comparison test and the ANOVA by using Software GraphPad Prism. The significance level was established at P< 0.05.

3. RESULTS:

3.1 Size of particles in Nanoemulsion:

The diameter of particles in Nanoemulsion crafted from Ethanolic bark extract of T. coriacea signifies their size distribution, crucial for stability and efficacy. Accurate sizing determines their potential in drug delivery or cosmetic applications, showcasing how the Nanoemulsion interacts within biological systems for enhanced performance and bioavailability. Particle size view of nanoemulsion given in figure no. 2.

Figure 2: Particle size view of Nanoemulsion in Electron Microscope.

Examining Nanoemulsion from Ethanolic bark extract of T. coriacea under an Electron Microscope reveals detailed particle size distribution and morphology. This high-resolution view elucidates the structure, shape, and uniformity of particles, vital for understanding their stability, interactions, and potential applications in pharmaceuticals or biotechnology with improved precision and insight.

3.2 Zeta potential:

The Zeta potential (mV) of Nanoemulsion derived from Ethanolic bark extract of T. coriacea reveals the electrostatic charge of particles. This charge affects stability and dispersion, influencing efficacy in applications like drug delivery. Precise Zeta potential measurement aids in understanding the Nanoemulsion's behavior, impacting its therapeutic potential. Zeta potential (mV) of particles of Nanoemulsion prepared from Ethanolic bark extract of T. coriacea given in fig. no. 3. The different diameter of particles in nanoemulsion given in figure no. 4.

Figure 3: Zeta potential (mV) of particles of Nanoemulsions prepared from Ethanolic bark extract of T. coriacea

Figure 4: Diameter of particles in Nanoemulsions prepared from Ethanolic bark extract of T. coriacea

3.3 Egg hatch test:

The data suggested the significant efficacy of the formulation. The four different dilutions (6.5%, 12.5%, 25% and 50% v/v) reduced the egg hatching of H. contortus by 55.24%, 65.62%, 88.26%, and 95.36%, respectively. A massive percentage of reduction (98.6%) was noted for the control (+) ve (Albendazole; 2mg/mL). The inhibitory effect of various dilutions of TcNano on egg hatch test is given in figure no. 5.

Figure 5: Inhibitory effect of various dilutions of TcNano on egg hatch test versus H. contortus from sheep (*p< 0.001)

3.4 Larval motility test:

The results of the larval movement investigations exhibited a significant effect by TcNano (fig. 2). The four dilutions (50%, 25%,12.5%, and 6.5%; v/v) reduced the egg hatching of H. contortus by 88.56%, 73.25%, 62.54%, and 51.26%, respectively. A massive percentage of reduction (88.56%) was noted for the control (Albendazole; 2mg/mL). The Inhibitory effect of various dilutions of TcNano on Larval motility test is given in figure no. 6.

Figure 6: Inhibitory effect of various dilutions of TcNano on Larval motility test versus H. contortus from sheep (*p< 0.001)

3.5 Adult worm motility test:

The mature worm testing revealed that the TcNano gave a paralysis time of 50.16±0.5 min. and a death time of 70.04±0.5 at 100mg/mL concentration. In 50mg/ml, it was 60.26±0.4 paralysis time and 78.53±0.2 min. death time observed, whereas in 10mg/ml, it was 94.52±0.9 paralysis time and 98.19±0.7 min. death time noted. The data on the efficacy of the anthelmintic activity of TcNano is given in Table No. 2.

Table 2: Anthelmintic activity of TcNano:

S. N.	Treatment	Conc. (mg/ml)	Paralysis Time (Min.)	Death Time (Min.)
1	Vehicle	-	-	-
2	Albendazole	10	27±0.5	30±0.8
5	TcNano	10	94.52±0.9	98.19±0.7
		50	60.26±0.4	78.53±0.2
		100	50.16±0.5	70.04±0.5

Each value characterizes by Mean ± SEM, (n=6), P<0.05; One-way ANOVA proceed by Dunnett’s multiple comparison tests.

3.6 LC-MS Interpretation of Phytoconstituents from Ethanolic Bark Extract of TC:

The analysis detected 227 compounds. Approximately 11 constituents demonstrated significant potential, including Glyceraldehyde, Phenylcoumarin, Cucurbitacin E, a surfactant compound, Norbuprenorphine-d3, Quinazolinamine, Triflupromazine, Flavanone, Mephenytoin, Methyl coumarin acetate, and Serpentine mineral cation. The LCMS chromatogram of ethanolic bark extract of T. coriacea is given in figure no. 7.

Figure 7: LCMS chromatogram of ethanolic bark extract of TC

4. DISCUSSION:

The genus ‘Terminalia’ is known to have huge range of significant chemical moiety. HPLC study confirmed the existence of ellagic acid, catechin, gallic acid and epicatechin, which are biomarkers, in the aqueous extract of Terminalia paniculata bark.⁴³ Terminalia arjuna contains a variety of phytoconstituents, including arjunic acid, arjungenin, arjunolic acid, arjunone, luteolin, arjunolone, gallic acid, ellagic acid, oligomeric proanthocyanidins and phytosterols. These compounds have a wide range of positive biological properties, such as anti-inflammatory, antioxidant, antimicrobial, antifeedant, and cardioprotective effects.⁴⁴ Various solvent solutions (including water, ethanol, methanol, chloroform, and ethyl-acetate) were used to extract different components from Teminalia chebula, such as fruits, seeds, galls, and barks. The extracts obtained contained bioactive chemicals like chebulinic acid, and chebulaginic acid, chebulic acid, which were found to have significant pharmacological activities.⁴⁵ The methanolic extract of Terminalia coriacea leaves showed the presence of flavonoidal components, which were identified in the extract through advanced analytical techniques. The mentioned chemicals consist of various compounds such as Quercetin-3-O-rutinoside, Myricetin hexoside, Luteolin-7-O-glucoside, Quercetin-3-O-glucoside, Isorhamnetin-3-O-rhamnosylglucoside, and Isorhamnetin-3-O-glucoside.⁴⁶

The advance formulation technique to incorporate the active ingredients in nano form delivery patter to enhance bioavailability of poor soluble content. Nanoemulsion have small droplet ranging from 20 to 200 nm which enhance the absorption to cells. Remarkable advancements have been achieved in the realm of nano-delivery systems, with the goal of enhancing the efficacy of traditional medications and mitigating their adverse effects. This is accomplished by developing intelligent delivery carriers with precise targeting abilities and regulated release mechanisms that are utilized in medical treatment.⁴⁷ People suffer substantial financial losses as a result of the neglected helminthic infections. The issue is further exacerbated by the emergence of drug resistance towards commonly prescribed anthelmintics, like triclabendazole. Thus, to develop future control tactics, it is crucial to investigate alternative approaches, such as those rooted in nanotechnology. The movement of mature worms was significantly impacted, and the treated showed signs of damage caused by reactive oxygen species (ROS) after being exposed to varying doses of silver nanoparticles in vitro. The worms that were treated exhibited noticeable DNA damage, along with compromised detoxification abilities and antioxidant system, in comparison to the control group.⁴⁸

Niclosamide is a widely recognized antihelminthic pharmaceutical that has been included as essential medication list due to its diverse pharmacological effects. Regrettably, the practical application of this substance is restricted because it is highly insoluble in water. We have successfully demonstrated the efficacy of a cost-effective lipid nanoparticle formulation of niclosamide that was created using only FDA-approved excipients.⁴⁹

At present, the available options for potent anthelmintic drugs are limited, and parasitic helminths are increasingly developing resistance to these medications. In addition, the scope of their activities is quite restricted. Explored the effectiveness of a colloidal nanosilver composition in eliminating parasites. It possesses the remarkable capability to eliminate all worms present in the experimental well. Research has shown that decreased levels of silver can negatively impact the protective cuticles of worms.⁵⁰ The complex interaction between enzymes and tannins, along with the chemical reactions between tannins and various non-protein organic nitrogen molecules, can lead to an increase or decrease in enzyme activity.⁵¹

A standardized and comprehensive method developed to weed out these substances that are enhancing the health of human, given the broad range of bioactive chemicals and the vast quantity of plant species⁵².

Natural product-derived metallic nano particles (MNPs) present a strong case for transforming antimicrobial tactics and offering a novel, effective, and sustainable means of preventing infectious illnesses and preserving public health ⁵³. This improved formulation has shown a notable reduction in helminths. The main goal of developing these formulations is to increase the efficacy of the constituents at the target site. The properties of the nanoformulations indicate a significant increase in bioavailability, leading to improved therapeutic outcomes. ⁵⁴. ⁵⁵ ⁵⁶

In vitro evaluation for the interdict of egg hatching⁵⁷, larva motility ⁵⁸, and adult worm movement test ⁵⁹ are generally used in parasitology to exploring of recent anthelmintic drugs⁶⁰. The convenience of these evaluations is that constituents or extracts to be assayed are directly associated with the several stages of the parasite. Thus far, in vitro investigation before in vivo evaluation has been proven to be an enlightened and experimental strategy; hence they are economically worth entirely. However, the constituents show an effect in vitro assay will reveal a response similar in vivo⁶¹ ⁶².

LCMS analysis is the chromatographic investigation to screen the responsible phytoconstituents.⁶³ To accurately quantify and identify the wide range of chemical constituents in mixture. ⁶⁴It has separation capabilities with sensitive detection allowing precise characterization of active compounds.⁶⁵ The analysis revealed the phytoconstituents such as Flavanone, Glyceraldehyde, Cucurbitacin E, Quinazolinamine, Phenylcoumarin, Norbuprenorphine-d3, Triflupromazine, Mephenytoin, Methyl coumarin acetate, and Serpentine. These belongs to alkaloids, flavonoids, triterpenoids and phenolic compounds which has reported to have helminthic efficacy⁶⁶.

The Terminalia genus plant bark part is also known for having tannin content and can be responsible for having anthelmintic activity. Therefore, the bark is selected for the study. The TcNano revealed significant anthelmintic properties on different selected eggs, larvae, and worms. TcNano has shown significant paralysis and death of adult earthworms compared to standard at the high dose of 10 mg/mL. This was also observed in the egg hatch and larval motility investigation compared to Albendazole at the higher amount of 2mg/mL.

Hence, in vitro results from the formulation TcNano on the model used suggested the significance of its efficacy. It is worth mentioning that the similar formulations showed significant anthelmintic effectiveness, similar to the positive control in the investigation of adult worms ⁵⁵. The LCMS investigation revealed the presence of significant constituents to support the activity.

Furthermore, it is imperative to comment that the equivalent nanoemulsion revealed a anthelmintic potency which is statistically significant, against the parasite's life cycle, such as eggs, larvae, and worms.⁵⁶.

There is a probability that alkaloids, tannins and phenolic compound contained in the TcNano produce activity. In another investigation, polyphenols exhibited to have anthelmintic effects against Nippostrongylus brasiliensis⁶⁷. Because tannins can bond with free proteins in the host animal's digestive system, they may also have an anthelmintic impact⁶⁸. Alternatively, a glycoprotein on the parasite's epidermis ⁶⁹ leads to death. According to several writers, providing more digestible protein enhances sheep's resilience and tolerance to gastrointestinal nematodes⁷⁰. Animals receive and absorb huge digestible protein from vegetation that contains tannin⁷¹. This is accomplished by tannins in the rumen forming protein complexes that later dissolve at low-level pH in the abomasum to discharge additional protein for ruminant animals' small intestines to use for metabolism⁷². In addition, the pre-parasitic phases of helminths are directly impacted by tannins or their metabolites. Essential oils, flavonoids, and terpenoids are phytochemicals with an anthelmintic effect ⁷³ ⁷⁴ ⁷⁵.

The summary of the mechanism is given in figure number 8.

5. CONCLUSION:

From the above investigation, it can be revealed that the TcNano shows significant results. This genus and plants have the traditional background to kill the different types of worms. The plant screened for in vitro helminthic activity in two other worms was near to standards. The chromatographic investigation (LCMS analysis) supported the significant efficacy of the formulation. These effects are because of alkaloids, tannins, flavonoids, and phenolic compounds. The plant part may be further investigated to isolate the individual chemical moiety responsible for the anthelminthic activity.

6. ACKNOWLEDGEMENT:

The authors want to acknowledge the GSP, GITAM Deemed University for providing facility to conduct the study and Vishakhapatnam campus for LCMS analysis. Authors also thank to Datta Meghe College of Pharmacy for providing software facilities.

7. DATA AVAILABILITY STATEMENT:

Data are contained within the article and Supplementary Materials.

8. CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

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