Invitro Evaluation of the Anti Helminthic Activity of Selected Eritrean Traditional Medicinal Plants

Human kind has been exposed to infection by microorganism since before the down of recorded history [1]. Diseases caused by intestinal parasites (helimentic) pose a great threat to health and contribute to the prevalence of malnutrition, anaemia and eosinophilia [2]. All cultures have folk medicine traditions that include the use of plants and plant products [3]. Traditional medicinal plants are linked to traditional culture with traditional healers and herbalists acting as important stake holders [3]. The plants are known torpor idea rich source of botanical and the lminthics. It is estimated that two thirds of the world population still rely on traditional medical remedies, mainly plants, because of limited availability and affordability of pharmaceutical medicines [4]. Today more than 70% of the people in Africa refer to traditional healers concerning health issues [4]. The world health organization (WHO) encourages the inclusion of her bal medicines of proven safety and efficacy in the health care program of developing countries because of the great potential they hold in combating various diseases [4]. Thus, limitations of reliance on chemo therapy are the threat of parasites developing resistance to drug treatment, the cost of drugs for people in developing countries and for some helminthes, lack of efficacy of current available drugs [5]. Therefore, novel and complementary helminthes control options are urgently needed. Taget esminutaItisa strongly scented annual herb with stiff erect stems up to 2m tall. Leaves stalked, opposite, light green, 7-15cm long, pinnately dissected into 4-6 pairs of pinnate. Secretions from roots are effective against nematodes, worms, insects, fungi and perennial weeds [6]. It has antimicrobial, antiphlogistic, sedative, fungicide and hypertensive properties [6]. Moringa Olifera an often referred to as the “miracletree” because of its uniquely diverse array of nutritional, medicinal, and purifying properties, It is a small or medium sized tree, fast growing deciduous or sharp tree that reaches 12min height when mature [6]. It’s known to have anti-diabetic, anti-hypertensive, antiinflammatory, anti-microbial, anti-viral, anti-parasitic, anti-tumor and anti-aging activities [6]. Abstract


Introduction
Human kind has been exposed to infection by microorganism since before the down of recorded history [1]. Diseases caused by intestinal parasites (helimentic) pose a great threat to health and contribute to the prevalence of malnutrition, anaemia and eosinophilia [2]. All cultures have folk medicine traditions that include the use of plants and plant products [3]. Traditional medicinal plants are linked to traditional culture with traditional healers and herbalists acting as important stake holders [3]. The plants are known torpor idea rich source of botanical and the lminthics. It is estimated that two thirds of the world population still rely on traditional medical remedies, mainly plants, because of limited availability and affordability of pharmaceutical medicines [4]. Today more than 70% of the people in Africa refer to traditional healers concerning health issues [4]. The world health organization (WHO) encourages the inclusion of her bal medicines of proven safety and efficacy in the health care program of developing countries because of the great potential they hold in combating various diseases [4].
Thus, limitations of reliance on chemo therapy are the threat of parasites developing resistance to drug treatment, the cost of drugs for people in developing countries and for some helminthes, lack of efficacy of current available drugs [5]. Therefore, novel and complementary helminthes control options are urgently needed. Taget esminutaItisa strongly scented annual herb with stiff erect stems up to 2m tall. Leaves stalked, opposite, light green, 7-15cm Cite this article: Yohana

Methods and Materials Used Collection and Identification of Earth worms
The assay was performed on adult earthworm (Pheritimaposthuma) owing to it's an atomically and physiological resemblance with the intestinal round worm parasites of human beings. Easy availability of earth worms and free-living lifestyle prompts their extensive use for preliminary in-vitro evaluation of anthelminthic compounds. Earthworms were collected from the water-logged soil besides Expo ground and were identified by an anatomist. All earthworms were of approximately equal size (15cm long).

Standards
The anthelminthic activity was carried out on freshly collected adult earthworms (Pheretimapostuma). The anti-parasitic activity was carried as per method of Ajaiyeoba et al. [7]. The earthworms were similar in length and width. They were washed with normal salineto remove soil, dust and mud from them and were put in petri-dish. Each Petri dish had three worms of approximately equal size ( Figure 1). Three different concentrations of 10mg/ml, 50mg/ ml and100mg/ml of each plant extract were used. The solutions were introduced into the respective petri-dishes. At the same time, positive (Mebendazole, 20mg/ml) and negative control (normal saline) were carried out.

Chemicals
The two plants were subjected to cold extraction using aqueous ethanol, Petroleum ether, Chloroform; Methanol and percentage yield was obtained.

Extraction method
The plant materials were washed under running tap water to remove mud and dust. The samples were air-dried for 7days and were well pulverized into a fine powder with a mixer grinder. The powder was stored in air tight aseptic containers (28ºC±2) for subsequent use. Weighed material (50grameach) was placed in conical flask containing the different solvents and then placed on an electronics haker for 48hours at the speed of 100rpm. The fluids were then filtered using what man No1 filter paper. The extracts were rotary dried to obtain the concentrate. It was then kept in the fridge (4ºC) prior to use.

Quality Control
All the equipments used for bioassay were auto calved/heat sterilized. Thepetri-platesand beakers prepared were auto calved at 121oc for 30minutes along with Bowiedick autoclave tape. During the analytical process Mebendazole as positive control and normal saline as negative control was used.

Histo-Pathological Studies
Histo-pathological slides were made from the treated earthworms with plant extracts. Sections from the earthworms in portions before and after the clitellum were selected. Fixations of these portions were made by 10% form alin for 24Hrs. These fixated pieces were put into the processing machine (alcohol, xyleneand Wax). They were further directed to embedding and moulding in an embedding medium causing them to solidify. The embedded portions were refrigerated, and the cooled portions were cut by amicrotome to 5micron thickness and were transferred to water (150C). Slides were made and transferred to oven with axylenecoat. The prepared slides were examined by apathologist. Finally, different pictures of the slides were taken and comparison with the control was made.

Statistical Analysis
The precision of this test methodology was determined by statistical analysis of all results on paralysis (P) and/or death (D) time. Repeatability was considered as the difference between successive results obtained by the same technician under constant conditions on identical worms, in the correct and standard procedure of the test methodology. Reproducibility was considered as the difference between two independent results obtained by different technicians working in different laboratories on identical test worms in the correct and standard procedure of the test methodology.

Results and Discussion
The highest yield percentage (22%) was obtained with aqueous ethanol and chloroform solvents from Moringa Olifera leaves. While the lowest yield percentage (7%) was obtained from the fruits of M.o lifera by using methanol. Similarly highest yield percentage (18%) was obtained with aqueous ethanol in case of T.minuta leaves and the lowest yield (8%) was obtained with chloroform and petroleum ether ( Table 1). The aqueous ethanol extracts of Targets minuta showed paralysis and death of the earthworms at 17±2.9min and 35±0.9min respectively at highest concentration of 100mg/ml and 32±1min and 210±3.6min respectively upon treatment with 10mg/ml. Results are found to bedose dependent with increasing concentration.   Data is an average of two experiments consisting three earthworms in one plate.
All the values represent Mean±S.D; n=3 in each group.
The death time of earthworm when treated with petroleum ether extract was 50±2.78min, 60±1.58min and 90±4min respectively using 100mg/ml,50mg/ml,10mg/ml concentrations, which was observed to have the longest death time as the concentration of plant extract was less than the two higher concentrations. Chloroform extract of Taget esminuta showed the most remarkable activity and paralyzed the earthworms within 55±0.6sec using 100mg/ mlof plant concentration, soit can be considered as most potent extract of the plant. The fourth solvent used for the extraction of Tagetes diminuta was methanol. It paralyzed the earthworms at 13±2.5min, 9±2.3min and 7±1.5min using the 10mg/ml,50mg/ml and 100mg/ml concentration while the death time was recorded 43±2.26min, 19±3.2min, 12±2.7min for the three concentrations (10mg/ml,50mg/ml and(100mg/ml) ( Table 2).
Aqueous ethanolic (70%ethanol) extract of Moringao lif era showed a variable effect when three different concentrations of (10mg/ml, 50mg/ml, 100mg/ml) were used. The highest concentration (100mg/ml) resulted in death of earth worms at 13±1.2min, whereas at the lowest concentration (10mg/ ml) death was observed at35±1.25mins (Table 2). In the case of Petroleum ether, paralysis of the earth worms was observed in12±2minfor the lowest concentration and 6±1minfor the highest concentration. Death time with petroleum the extract was recorded at 50±4.24min, 38±1.6min, and 20±3min at 10mg/ml, 50mg/ml and 100mg/ml concentration respectively. Chloroform extract also showed good and the lminthic activity. Results clearly indicated that with increased concentration, the effect also increased. Among all the solvents used, Methanol showed the third better activity as the paralysis and death time at the highest concentration wasobservedat4±0.8minand 28±2minrespectively ( Table 2).
Aqueous-ethanol and methanol were used for extraction of Moringao lifera fruits. Aqueous-ethanol extract with Concentration of 10mg/ml, 50mg/ml and 100mg/ml showed a paralysis time in 4±1.8min, 2±1.2min and 55±0.4sec respectively, whereas the death

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time were recorded at 8±2.1min with the highest concentration and 45±2.7 min with the lowest concentration. For the methanol extract, paralysis and death time of the earthworms were recorded 50±0.5sec and 4±2.2minat the highest concentrations respectively. Both the leaf and fruit extract of M.oliefera have homicidal effect. Also the fruit extract of M.olifera has still better damaging effect than that of leaf extract of this plant. The effect of extracts of Moringa Olifera leaves on the paralysis of the worm, according to the results (Table 3) may be indicated as Aqueous ethanol >Methanol >Petroleum Ether>Chloroform. Data is an average of two experiments consisting three earthworms in one plate.
All the values represent Mean±S.D; n=3 in each group.
The combination of the M.olifera (fruit and leaves) and T. Minuta (leaves) showed paralysis at 15±3min with the lowest concentration of Aqueous ethanolic extract and at 3±2min with the highest concentration, where as death time was recorded at 40±3.6min and 12±3min respectively. The 50mg/ml concentrations paralyzed the earthworms within 8±1min and killed them in 20±4. 2min. Theplants' extractives showed the lminthic activity and were dose-dependent that increase in concentration of plant showed increased destruction on earthworms. In the combination, the antihelminthic activity of aqueous ethanolic extracts of M.oliefera (fruit and leaves) was reduced when tested in combination with T. Minutaal though acleardose-dependent effect indicating increased activity with increased concentration has been found ( Figure 2). for death of worms. Albendazole was used as a reference standard and saline as a control group. The ethanol extract showed more significant effect on paralyzing the worms, in terms of paralysis time, at every concentration compared to that of ethyl acetate, water, chloroform, hexane extracts. In particular the ethanol extract exhibited an increased paralytic as well as helimentic effect overall bendazol eat the given experimental concentrations [8].
In another study the root part of Capparisdecidua Edge was evaluated for the an the lminthic activity of the plant at 10, 50 and 100mg\ml using ethanol as the main extraction solvent against Indian earthworm Pheritimaposthuma. The activity was found dose dependent compared with the standard drugusedat10mg/ mlas activity increased with increase concentration of plant [9]. A study conducted on the anti-helminthic activity of the drupe extracts of MeliaazedarachL. (Meliaceae) growing in Argentina against a tape worm and an earthworm, showed to be better than the standard piperazine phosphate, which is used in the treatment of Cestoda infections as it showed mean paralysis and death time value at 32min and 68min using 70%Ethanol at high (80mg\ml) and low (20mg\ml) concentration [10].
In the present study when the fruits of Moringa Olifera were extracted in aqueous ethanol (70%) and methanol, both showed excellent an lminthic activity. The earthworms were paralyzed in 55±0.8sec and 50±0.5sec in aqueous ethanol (70%) and methanol extracts respectively at100mg/ml while death was caused at 8±2.1min with aqueous ethanol extract and 4±2.2min with methanol extract at same concentration. Several studies showed that extracts from plants grown to serve as human food for example: coconut, onion and garlic, annan as, chicory have high Anti Helmentic potential against intestinal nematodes, cestodes, and trematodes [11].

Histo pathological studies
Effect of treatment with Tagetes diminuta extract: Histo pathological observations revealed that treatment of earthworms with leaf extract of T.minuta at 100mg/ml (Chloroform extract) were effective. The epidermis and the overlying cuticle were damaged; epidermal cells appeared to be bulged. There was reduction in the height of circular muscle layer (CML) and longitudinal muscle layer (LML) in comparison to the control (saline).The coelomicsepta and gut epithelium appeared to be distorted. The vasculature of the body and the VNC were found to be intact. The histopathological damages due to these extracted plants were compared to that of control animal picture (Figure 3).

Effect of treatment with Moringa Olifera extracts
Histo pathological studies revealed that animals treated with leaf extracts of M.olifera (100 mg/mlof70%-ethanol) showed damaging effect on their body wall as well as visceral organs. The epiderm is of the body wall was more or less completely destroyed. Epidermal cells were variously damaged, although cuticle, the upper most lining of the body wall was less affected. The musculature of the body wall has also showed reduction. The CML and LML appeared to be reduced in their height in comparison to the selayer of normal control animals. The visceral organs such as coelomicsepta (CS) and gutepi the lium were completely dismantled. However, the dorsal and ventral blood vessels were found to be less affected. The ventralnervecord (VNC) on the other hand was intact (Figure 4). It is worth to mention here that fruit extract of same plant was more effective than its leaf extract ( Figure 5)

Effect of treatment with combined extracts
The histopathological studies pertaining to effect of combined extracts of M.olifera and T. Minuta on the experimental worm revealed that there was no remarkable damage on the body wall. However, the epidermal cells seemed to be partly damaged. The cell size was relatively enlarged and some of the cells were completely damaged. The height of CML and LML was also reduced as it was observed in animals treated with leaf extracts of both plants. The visceral organs such as coelomic septa and gut were also damaged to a large extent. Vasculature and nervous tissues, especially VNC do not seem to be much affected ( Figure 6). Animals treated with reference drug did not show any remarkable damaging effect on the tissues.

Conclusion
The present study was an attempt to conduct in vitro antihelminthic activity of selected Eritrie an medicinal plant to substantiate the folk claim. This study reveals new scientific information about the leaf extracts of Taget esminuta and leaf and fruits extractives of Moringa Olifera rain regard to a the lminthic activity. The plants' extractives showed a the lminthic activity and were dose-dependent that increase in concentration of plant showed increased activity on earthworms.
Fore going observation on the effect of the extracts of the two selected plants lead us to reach the conclusion that both the leaf and fruit extract of M.olifera as well as the leaf extract of T.Minuta have wormicidal effect. Also the fruit extract of M.olifera has still better damaging effect than that of leaf extract of this plant. Present observation suggests that these herbal materials should be regularly used to get protected from the ill effects of the worms because these herbs have worm icidal effects. The exact mechanism of action of these plants is not known. However, it is apparent that these extracts cause damage to tissues of worms. Since these plant extracts have quick paralytic effect, it is advisable to make regular use of such herbs. Because once the worm is paralyzed, there is likely hood of worms to be easily flushed out from the body.
To conclude, in future studies, there is need for thorough phyto chemical, clinical and possible studies on molecular mechanism of action. At the same time efforts should be made to standardize the plant extracts with good antihelminthic activity and formulate best alternative herbal preparations to replace or complement the synthetic drugs which are currently in use (Table1)