In-vitro study of antiproliferative and anthelminthic property of medicinal plants of Kokrajhar, Assam


 People living in far-flung areas of the world, especially ethnic tribal people practice traditional medicine as the first choice of disease treatment. The present study investigates the antioxidant, cytotoxicity, and anthelmintic activity of four medicinal plants traditionally used by tribal communities of Bodoland Region of Assam. Total phenolic and flavonoid content was estimated following spectrophotometry method. Antioxidant activity was measured by total antioxidant assay, FRAP, DPPH, ABTS, and TBARS assay. Antiproliferative and apoptosis-inducing activity of plants were carried out in DL cells. Cells were treated for 24 h with different doses of plant extracts. Furthermore, anthelmintic study was carried out by treating the helminth parasite at different doses of plant extracts. Phytochemical and antioxidant study showed rich TPC, TFC, and free radical scavenging activity in H. japonicum and H. sibthorpioides. Both the antiproliferative and anthelmintic activity showed a dose-dependent efficacy in all the plants. H. japonicum showed the strongest anthelmintic activity with LC50 212.78 µg/mL followed by H. sibthorpioides (5.36 mg/mL), C. halicacabum (13.40 mg/mL), and A. scholaris (18.40 mg/mL). On the other hand, H. sibthorpioides showed stronger antiproliferative and apoptosis-inducing activity compared to other plants. The study observed a positive correlation between the antioxidant property and antiproliferative and anthelmintic activities of the plants. We, therefore, conclude that the secondary metabolites along with antioxidant molecules may have combined effects contributing the antiproliferative and anthelmintic activity of the plants.


Introduction
Helminths are a group of eukaryotic organisms that affects millions of people worldwide and also cause severe economic loss to the livestock industry (Qamar et al. 2011). Among the major helminthiasis, soiltransmitted helminthiasis and schistosomiasis represents more than 1 billion people worldwide, mainly in poor economic countries of the world (James et al. 2018). It has been estimated that the mass deworming program of helminth infestation cost about US$300 million dollars annually while the cost of treatment via screening programs would likely be US$2 billion annually (UCNTD 2014). Since several decades, the use of commercial anthelmintic such as albendazole, mebendazole, benzimidazole, etc. is the most common practice of controlling helminthiasis. However, there is a growing report of drug resistance in helminth parasites leading to ineffective controlling of helminthiasis through drug administration (Mphahlele et al. 2019). Plants are known for its rich phytochemicals and medicinal values and are therefore investigated for its anthelmintic properties throughout the world as an alternative to the existing commercial drugs. Several medicinal plants around the world are screened for their anthelmintic property by many researchers (Roy and Swargiary 2009;Carvalho et al. 2012;Tandon and Das 2018).
Assam is one of the north eastern states of India rich with ora and fauna. People living in this part of India, especially ethnic tribal groups perform several traditional practices to control common ailments including helminth infestation. Several plants are documented by many authors from this part of India (Swargiary et al. 2017;Panda et al. 2018; Daimari et al. 2019). In our earlier studies we have reported several medicinal plants traditionally practiced as antidiabetic and anthelmintic agents by the tribal communities of Kokrajhar, Chirang, Baksa, and Udalguri district of Assam (Swargiary et al. 2016;Swargiary et al. 2019a, b;Swargiary et al. 2020

Preparation of crude plant extracts and solvent fractions
Bark of A. scholaris, and leaves of C. halicacabum, H. sibthorpioides, and H. japonicum were washed properly to remove dart particles and processed for methanolic crude extraction following the method described in our earlier publication (Swargiary et al. 2016). Brie y, plant parts were dried completely in hot-air oven below 50°C. Dried plants were powdered using mixture grinder. Plant powder was mixed in 80% methanol (1:5, w/v) and kept for 72 h. After that the solutions were ltered with Whatman lter paper No. 1 and the ltrate obtained is dried in a rotary evaporator. The solid material felt is collected as crude extract and kept in -20°C till further use.

Phytochemical and Antioxidant Study
Total phenolic content (TPC) The total phenolic was estimated using Folin-Ciocalteu reagent (Iloki-Assanga et al. 2013). TPC was calculated from a calibration curve of gallic acid and results expressed as µg gallic acid equivalent (µgGAE)/mg plant extract.

Total avonoid content (TFC)
The avonoid content was determined following the method of Ordonez et al. (2006). TFC was calculated from the standard curve of quercetin and the values were expressed as microgram quercetin equivalent (µgQE)/mg plant extract.

Antioxidant Study
Total antioxidant capacity (TAC) assay TAC of the plant extract was done by phosphomolybdate method using ammonium molybdate reagent (Huda-Faujan et al. 2009). TAC was expressed as µgAAE/mg plant extract.
Ferric reducing antioxidant power (FRAP) assay FRAP assay was performed following the method of Iloki-Assanga et al. (2015). The FRAP activity is compared with the standard ascorbic acid and values were expressed as μg Fe 2+ equivalent (μgFE)/mg plant extract.
Lipid peroxidation scavenging activity (Thiobarbituric acid reactive species) assay Lipid peroxidation inhibitory activity was studied following the modi ed thiobarbituric acid reactive species (TBARS) assay to measure the lipid peroxide formation using egg yolk homogenates as lipid-rich media (Okhawa et al. 1979). The coloration of the assay mixture was measured at 532 nm.

In-vitro anthelmintic bioassay
Live, adult trematode parasite, Paramphistomum sp. were collected from the rumen of cow from Kokrajhar town. In-vitro anthelmintic study was carried out following Belemlilga et al. (2016). Parasites collected in phosphate buffered saline (1xPBS, pH 7.4) were brought to the laboratory and allowed to acclimatize for 30 min at 37±1ºC. Next, the parasites were incubated in a series of extract concentrations. After 24 h of treatment mortality was noted and the lethal concentration at 50% mortality (LC 50 ) was calculated. Albendazole (ALB), a broad-spectrum anthelmintic drug was used as reference chemical. Control ukes were incubated in PBS alone. For each set of experiment three replicates (n = 3) were carried out.

Cell proliferation and apoptosis study
The antiproliferative and apoptosis-inducing activity of plant extracts was studied using Dalton's lymphoma (DL) cell line as described in our earlier publication (Swargiary et al. 2021). Cell proliferation was measured by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann 1983;Verma et al. 2013). In brief, the cells were treated with different doses (10-200 mg/ml) of plant extracts for 24 h in a 96-well plate. Next, 10 µl of MTT reagent was added and incubated for 4 h at 5% CO 2 and 95% air at 37ºC followed by addition of 100 µl DMSO. The colour developed was measured at 570 nm using Elisa Microplate Reader. Furthermore, for apoptosis study, both the control and plant extract treated cells were stained with acridine orange/ethidium bromide (AO/Eb) for 5 min in dark cold room (Squier and Cohen 2001). The cells were then thoroughly examined for three replicates under uorescence microscope and photographed. About 1000 cells were counted, and the percentage of apoptotic nucleus was determined based on differential staining pattern (red/green) of the nucleus.

Statistical analysis
All the statistical calculations were carried out in excel. LC 50 and IC 50 were calculated using OriginPro and SPSS software. All the experiments were carried out in triplicates (n = 3) and the results were represented as mean ± standard deviation (SD).

Plant extracts and phytochemical content
Plants are rich in phytochemicals and contain various primary and secondary metabolites. The medicinal properties of a plant are attributed to its secondary metabolites -phenolics, avonoid, etc. The TPC, TFC, FRAP and total antioxidant property of the plants are shown in Fig. 1

Antiproliferative and apoptosis study
The antiproliferative and apoptosis inducing properties of all the four plants are presented in Fig. 3. The study revealed a dose-dependent cytotoxicity effects of the plants against DL cell. Of the four plants, H. sibthorpioides showed strongest cytotoxic property. MTT assay showed that the % death of DL cells ranged from 23±20% to 58.67±4.73%, 17.33±1.53 to 43±4.36%, 13±2.64 to 27.67±3.05% and 3±1 to 15±3.61% for H. sibthorpioides, H. japonicum, A. scholaris, and C. halicacabum, respectively at the concentration range of 25 -200 mg/mL after 24 h treatment. Similar results were seen in the apoptosis study indicating the potency of plant extracts to induce apoptosis and cell death. At 200 mg/mL concentration of plant extract, the cell death was found to be 42.33±3.21%, 32.37±2.52%, 22±4% and 9.0±1.0% for H. sibthorpioides, H. japonicum, A. scholaris, and C. halicacabum, respectively. The study showed a positive and signi cant correlation (P≤0.05) between antiproliferative and apoptotic properties of the plants. Changes in the morphological features with apoptotic characteristics with red/orange nuclei, membrane blebbing, chromatin condensation, and formation of apoptotic bodies have been observed in DL cells treated with plant extracts and reference drug, cisplatin (Fig. 4). Control cells showed green nuclei with intact membrane. AO/Eb staining showed higher density of apoptotic cells in H. sibthorpioides treatment followed by H. japonicum, and A. scholaris. C. halicacabum extract showed the lowest apoptosis-inducing property cells.

Anthelmintic activity
The trematode parasite, Paramphistomum sp. when exposed to different concentrations of plant extract showed dose-dependent mortality after 24 h treatment. Fig. 5 showed the percent mortality of the parasite treated with different concentrations of extracts. H. japonicum showed the strongest parasite mortality followed by H. sibthorpioides, C. halicacabum, and A. scholaris. The plant also showed better activity compared to reference drug, albendazole. At highest dose of 2 mg/mL, H. japonicum showed almost 100% mortality in helminth parasites. Similarly, at 20 mg/mL plant of H. sibthorpioides and A. scholaris, helminth parasites showed almost 100% and 60% mortality. While, C. halicacabum attained almost 100% mortality at 40 mg/mL. The LC 50 value was found to be 212.87 µg/mL, 5.36 mg/mL, 13.40 mg/mL, and 18.40 mg/mL for H. japonicum, H. sibthorpioides, C. halicacabum, and A. scholaris, respectively. Similarly, the LC 50 value of reference drug, albendazole was found to be 3.69 mg/mL. The control, untreated parasite lived up to ~73 h.

Discussion
Free radicals are by-products of biological reactions which produce several health effects. Biomolecules, called antioxidants, can neutralise and scavenge those free radicals. Plants are known to contain strong antioxidant property due to its rich secondary metabolites. Polyphenolics and avonoids are among the most important phytochemicals with rich antioxidant property. Phytocompounds can provides health bene ts in many ways such as substrates for biochemical reactions, co-factors of enzymatic reactions, enzyme inhibitors or stimulators, scavengers of reactive or toxic chemicals, and many more (Dillard and German 2000). Plants, in addition to primary metabolites, also produce secondary metabolites that help in normal growth, development, and defence system of plants. Among all the different type of secondary metabolites, phenolics are the most important because of its promising antioxidant properties (Horwitt 1991). The antioxidant property of plants may be attributed to its innate ability to synthesize nonenzymatic antioxidants such as ascorbic acid, glutathione as well as secondary metabolites such as phenolic compounds. The present study revealed considerable amount of phenolics and avonoid content in all the four plants. Many ndings suggest that the high TPC and TFC can improve biochemical indices of oxidative damage (Sera ni et al. 1996;Stein et al. 1999). The continuous generation of free radicals may cause severe complication if in excess. To minimise such complications, intake of antioxidant is always bene cial. In the present study, H. japonicum, and H. sibthorpioides showed higher phenolic and avonoid contents among the four plants. Similar kind of study showed comparable data of phenolics and avonoid content (Irshad et al. 2012). The methanolic peel extract of Citrus grandis also showed comparable data with our ndings (Dibya et al. 2016). Like most of the other studies, we also found potent antioxidant property in all the plants. Statistical analysis revealed that the in vitro antioxidant study by TAC, FRAP, DPPH, ABTS and TBARS assays showed positive correlation to TPC and TFC which reinstate the function of phenolics as potent antioxidant molecule.
Growth and development of an organism depends on the proper regulation of cell division and death. Any deviations from the normal physiology of cell growth, division, and death leads to the development of disease and complications. Unregulated cell division and growth is an important characteristics of cancer disease (Wong 2011). Apoptosis is a cellular mechanism that causes normal cell death, also known as programmed cell death. Cancer cells are known to avoid apoptosis and normal cell death cascade leading to uncontrolled cell division. Because of its cellular importance, apoptosis-inducing drugs have been shown to be the centre of new anti-cancer therapy (Elmore 2007). Several studies have been carried out to explore the antiproliferative and apoptosis-inducing properties of several medicinal plants (Rais et al. 2019;Khurshid et al. 2020). Phytochemicals and bioactive compounds isolated from plants were also investigated for anti-cancer activity in numerous cell lines (Kamaruddin et al. 2019;Erdogan et al. 2020).
In the present study, H. sibthorpioides, and H. japonicum showed better antiproliferative and apoptosisinducing property compared to other plants. Both the plants showed high cell mortality (~50%) at the highest dose of plant extract. The various phytocompounds present in the plant extract and their synergistic effects may have contributed to the antiproliferative and apoptotic activity. It is reported from many studies that studies that the crude extracts of plants show lesser biological activity compared to the isolated compounds (Lowe et al. 2013;Alasmary et al. 2018;Swargiary et al. 2021). Cisplatin, the reference chemical showed a signi cant difference (at P≤0.05 level) in both antiproliferative and apoptotic inducing capacity compared to medicinal plants.
Helminth infestation is one of the 17 Neglected Tropical Diseases of the world (WHO 2010). According to recent publication India stand global leader in many of the NTDs including helminthiasis and other vector borne diseases (Hotez et al. 2018). Most of the time development of drug resistance by helminth parasites is recognised as the major hurdle in effective controlling of helminthiasis. As an alternative or supplementary to the existing system of synthetic medicines plants are regarded as an effective tool in dealing helminthiasis. In the present study, we have investigated the anthelmintic property of four medicinal plants consumed by the tribal groups of Bodoland Region of Assam. In a similar study, Kumar et al. (2013) established strong anthelmintic property of the bark extract of A. scholaris. Leaves of H. japonicum showed the strongest anthelmintic activity among all the plants while A. scholaris showed the weakest activity. H. japonicum is also known to contain several pharmacological properties such as anticancer, hepatoprotective, antiviral, and immune-boosting activity (Liu et al. 2014). Several other studies have reported the anthelmintic activity of many plants (Roy and Swargiary 2009;Irshad et al. 2010;Swargiary et al. 2017). Wahyuni et al. (2019) also reported in vivo anthelmintic activity of four plants belonging to Cassia sp. from Indonesia showing highest activity in Cassia surattensis. Citrus is an important fruit plant having tremendous medicinal property. Several species of citrus have been investigated for its therapeutic potential by many researchers. Accordingly, C. sinensis, C. medica and C. reticulata have been reported to possess anthelmintic property against Haemonchus contortus and P. posthuma (Gainza et al. 2015;Aryal et al. 2017). Studies have revealed positive relationship between the phenolics and pharmacological properties of the plants (Akkari et al. 2016). The phenolic compounds of plants can interfere with the oxidative phosphorylation pathway of helminth parasites leading to the inhibition of ATP synthesis and induce mortality (Athnasiadou et al. 2001). Studies also reported that the phenolic compounds can bind to glycoprotein on the cuticle of helminth parasites and causes death (Salhan et al. 2011). The present study also observed positive correlation between the antioxidant, antiproliferative, and anthelmintic activity of the plants. The present study observed positive correlation between antioxidant property, antiproliferative, and anthelmintic activity of the plants. The secondary metabolites responsible of high antioxidant property may also have contributed to strong antiproliferative and anthelmintic property of the plants in the present study. The cytotoxicity property of the plants is directly correlated to the mortality of cells. Higher the cytotoxicity property of plants higher cell death. The present study also revealed that higher the cytotoxicity of the plant stronger the anthelmintic activity.

Conclusions
The current study validates the traditional knowledge and faith of people practicing since ancient times. All the four plants showed promising antiproliferative and anthelmintic activity. The rich secondary metabolites present in the plant extract may have synergistic effect in contributing the cytotoxicity and anthelmintic property of the plants. H. sibthorpioides and H. japonicum showed promising antiproliferative and anthelmintic property. However, further phytochemical characterisation and in vivo bioassay need to be carried out to explore the bio-active compounds responsible for antiproliferative and anthelmintic activity of the plants. Apoptotic features of plant extract-treated cells observed under uorescence microscope after AO/Eb staining. Cisplatin is used as reference drug. Apoptotic cells are shown in red/orange nucleus, control cells showed green nucleus