تقدير مدى فاعلية بعض أنواع نبات الشيح المصرى كمبيد رخوى واستخدامها كعلاج لمرض البلهارسيا المعوية

                                                "المستخلص

أجريت هذه الدراسة بهدف معرفة تأثير نبات ارتيمسيا أبثنثيوم (الشيح الخرساني) وارتميسيا انكولتا (الشيح البلدي) على قواقع بيومفلاريا الكسندرينا العائل الوسيط للبلهارسيا المعوية في مصر. وكذلك تأثيرها على البويضات والأطوار الحرة لطفيلي البلهارسيا (الميراسيديا و السركاريا). وأيضا دراسة تأثير كل من النباتين كعلاج لمرض البلهارسيا المعوية. واستخدام عقارالبرازيكوانتيل كعلاج لمرض البلهارسيا المعوية.

وقد أوضحت نتائج الدراسة أن المستخلص النباتي المغلي لكل من النباتين ليس له تأثير كمبيد رخوي على قواقع بيومفلاريا الكسندرينا عند تعرضها إلى 1000 جزء في المليون لمدة 24 ساعة. المستخلص الكحولي لكل من النباتين له تأثير سام على قواقع بيومفلاريا حيث كان التركيز المميت لـ 90% من القواقع 250 و 400 جزء في المليون على الترتيب بعد التعرض 24 ساعة. ووجد أن التركيز المميت لـ90% من القواقع من نباتي الشيح الخرساني والشيح البلدي قد أباد 82% و 78% من بويضات البلهارسيا على التوالي خلال 15 دقيقة من التعرض. والأطوار الحرة لطفيلي البلهارسيا المعوية (الميراسيديا والسركاريا) قد ماتت بعد 45 دقيقة و30 دقيقة على التوالي عند التعريض للتركيز السابق لكل من النباتين.

وفى هذه الدراسة تم استخدام ثلاث جرعات مختلفة من المستخلص الكحولى لكل من النباتين (500 ، 700 ، 800 مليجرام / كجم من وزن الفأر). وقسمت الفئران إلى خمس مجموعات المجموعة الأولى أعطيت العلاج بعد 7 أسا بيع من العدوى و المجموعة الثانية أعطيت ثلاث جرعات بعد الأسبوع الأول والثاني والثالث من العدوى و المجموعة الثالثة أعطيت جرعة قبل العدوى بيوم وجرعتين بعد الأسبوع الأول والثاني بعد العدوى والمجموعة الرابعة أعطيت جرعة بعد24 ساعة من العدوى ثم جرعتين بعد الأسبوع الأول والثاني بينما المجموعة الخامسة أعطيت جرعة 800 مجم / كجم من وزن الفار من نبات ارتميسيا انكولتا بعد 7 ، 8 ، 9 أسابيع من العدوى. ومجموعة أخرى تم علاجها بعقار البرازيكوانتيل (الجرعة 500 مجم / كجم من وزن الفار) لمدة يومين متتاليين بعد 7 أسابيع من العدوى.

ولقد أوضحت النتائج أن العلاج بمستخلص نباتي الشيح الخرساني والشيح البلدي أدى إلى نقص في عدد ديدان البلهارسيا المعوية في كل المجموعات ووجد أن نسبة النقص في عدد الديدان في حالة العلاج بالجرعة 800 ملجم / كجم من وزن الفار من مستخلص النباتين بعد 7 أسابيع من العدوى كانت 39.05%  و 38.7 % للنباتين على التوالي. بينما كانت84.2% و82.6%  في المجموعة المعالجة بنفس التركيز جرعة قبل 24 ساعة من العدوى وجرعتين على التوالي بعد الأسبوع الأول والثاني من العدوى.

وقد لوحظ أيضا أن العلاج بمستخلص كل من النباتين قد احدث نقصا إحصائيا في عدد البويضات في أنسجة الكبد والأمعاء. كما حدث انخفاض إحصائي واضح في عدد البويضات في البراز.

أن العلاج بمستخلصي النباتين الشيح الخرساني والشيح البلدي (الجرعة 800 ملجم / كجم من وزن الفار) قبل 24 ساعة من العدوى وجرعتين على التوالي بعد الأسبوع الأول والثاني من العدوى قد أدى إلى انخفاض في حجم الأورام الحبيبية وكان أقصى تأثير لوحظ عند العلاج بمستخلصي النباتين 24 ساعة قبل العدوى ثم بعد 7 و 15 يوما من العدوى (44.55% ، 40.37% على التوالي) ولم يحدث تحسن في الشكل الهستولوجى لأنسجة الكبد.

وعند العلاج بالبرازيكوانتيل (جرعة 500 ملجم / كجم من وزن الفار لمدة يومين متتاليين بعد 7 أسابيع من العدوى)كانت نسبة النقص في عدد ديدان البلهارسيا المعوية 96.4%.  وقد حدث نقص في عدد البويضات في أنسجة الكبد والأمعاء وفى عدد البويضات في البراز. وقد احدث العلاج بالبرازيكوانتيل انخفاض كبير في حجم الأورام الحبيبية وكانت lobular مع تحسن في الشكل الهستولوجى لأنسجة الكبد.

ومن هذه الدراسة نستنتج أن المعاملة بمستخلص  نبات الأرتميسيا يعطى تأثير عالي عند إعطاء جرعات متتالية من النبات في المراحل الأولى من المرض.

SUMMARY

 

Schistosomiasis is an endemic parasitic disease in several parts of the world, and is one of the most important environmental diseases in Egypt, thus considerable attention has been focused on its control by various means.

The current status of schistosomiasis depends on the existence of an intermediate host (snails) and the definitive host (human).

Although chemicals have been screened for molluscicidal activity since, most have proved deleterious to the environment and today only one synthetic molluscicide (Bayluscide) is commercially available.

The aggressive use praziquantel (PZQ) to combat schistosomiasis in Egypt raises concern about the possible emergence of resistant strains.

 

The present study is designed to evaluate the effect of an Egyptian strain of Artemisia as a molluscicidal and schistosomicidal acceptable with no apparent side effects.

The experimental design included two main experiments:

Experiment (1):

The present study was designed to evaluate the molluscicidal activity of the plant powder suspension of boiled water and the ethanol extracts of the dry powder of the two plants namely Artemisia inculta (A. inculta) and Artemisia absinthium (A. absinthium) from family Compositae, against Biomphalaria alexandrina snails, thereafter, revealing which is more effective of these plant species. The effect of the extracts of the two plant species was determined against the ova and the living larval stages of S. mansoni (miracidia and cercariae).

 

Experiment (2):

This was designed to study the effect of A. inculta or A. absinthium extracts on schistosomiasis. Five hundred and seventy male mice weighing (18-22 gm) and about 8 weeks old, were divided into:

A. Non-infected groups:

1-            Control mice: Including 10 normal mice.

2-            Treated control: Including 20 normal mice of which, 10 mice were treated with A. inculta and the remaining 10 mice were treated with A. absinthium (in single oral dose of 800 mg/kg body weight), and they were sacrificed two weeks later.

B- infected groups:

Including 340 mice. All animals were infected with 100 S. mansoni cercariae. They were divided into 6 main groups and hence 6 experiments:

-              Group I (control): including 20 infected male mice, for each experiment.

-              Group II: including 160 male mice. Eighty mice received treatment with A. absinthium in a dose of 500 mg/kg body weight and the other eighty mice received A. inculta in a dose of 500 mg/kg b.w. This group is further subdivided into four subgroups according to the duration of treatment:

1-            Subgroup 1: including 40 male mice (20 for each plant), treated with the above mentioned doses 7 weeks post infection and were sacrificed 9 weeks post infection.

2-            Subgroup 2: including 40 male mice (20 for each plant), treated with the above mentioned doses 7, 14 and 21 days post infection and were sacrificed 9 weeks post infection.

3-            Subgroup 3: including 40 male mice (20 for each plant), treated with the above mentioned doses one day pre-infection then 1 & 2 weeks post infection and were sacrificed 9 weeks post infection.

4-            Subgroup 4: including 40 male mice (20 for each plant), treated with the above mentioned doses one day post infection and 1 & 2 weeks post infection and were sacrificed 9 weeks post infection.

-              Group III: including 160 male mice received treatment with A. absinthium and A. inculta in a dose of 700 mg/kg b.w. and subdivided into four subgroups treated as mentioned previously in group II.

-              Group IV: including 160 male mice received treatment with A. absinthium and A. inculta in a dose of 800 mg/kg b.w. and subdivided into four subgroups treated as mentioned previously in group II.

-              Group V: including 20 male mice treated with A. inculta (in a dose of 800 mg/kg b.w.) 7, 8 and 9 weeks post infection and were sacrificed 10 weeks post infection.

-              Group VI: including 20 mice, each mouse was infected with 100 S. mansoni cercariae, 7 weeks later they were treated with praziquantel (PZQ) for two successive days in a dose of 500 mg/kg b.w. Fifteen days later, scarification was done.

The following parameters were studied:

1-            Parasitological parameters:

-              Worm load and distribution

-              Tissue egg load

-              Oogram pattern

-              Ova count in stool using kato technique

2-            Histopathological study:

-              Measurment of liver granuloma diameter and recording their number.

-              Study of liver histopathological changes in infected and treated mice in the different groups.

 

The study results are summarized as follow:

1-            The water suspensions of the boiled powder of the two plants were not lethal against B. alexandrina snails with 1000 ppm after exposure for 24 hours.

2-            The ethanol extracts of A. absinthium and A. inculta were lethal to B. alexandrina snails. Data indicated that the LC50 and LC90 values were 102 ppm and 250 ppm for A. absinthium and 180 ppm and 400 ppm for A. inculta, respectively after exposure for 24 hours. A. absinthium proved to have the superior molluscicidal activity against B. alexandrina snails than A. inculta. The ova of S. mansoni were dead after 15 minutes of exposure to LC90 of the two plant species (being 82% and 78%, respectively).

3-            The free living larval stages of S. mansoni, (miracidia and cercariae), were dead after 45 and 30 minutes of exposure to the LC90 of A. inculta and A. absinthium, respectively.

In the second experiment of this study, the data collected after sacrifice of the animals treated with the extracts of A. absinthium or A. inculta showed a significant reduction in the number of S. mansoni worm load.

Treatment with the extract of the two plants in a dose of 500 mg/kg b.w. 7 weeks post infection showed that the percentage reduction of the mean number of S. mansoni worms after administration of A. absinthium or A. inculta was (28.17% and 26.7%, respectively).

In this study, treatment with A. absinthium or A. inculta (in a dose of 700 mg/kg or 800 mg/kg b.w.) showed no differences at the different periods of exposure. The group treated 7 weeks post infection showed a percentage reduction of the mean number of both of A. absinthium or A. inculta was 38.7% and 39.5%, respectively.

Treatment with the same dose (500 mg/kg b.w.) initiated 7, 15 and 21 days post infection showed a highly significant percentage reduction in the mean number of S. mansoni worms (60.30% and 52.76% for A. absinthium or A. inculta, respectively).

In this work, treatment with A. absinthium or A. inculta by the same dose (500 mg/kg b.w.) at 24 hours pre-infection and 7, 15 days post infection resulted in a highly significant reduction in the percentage of the mean number of worms (71.1% and 62.8%, respectively).

In the group treated with A. absinthium or A. inculta by the previous dose 24 hours and 7 and 15 days post infection, the results of worm reduction were 65.43% and 57.81%, respectively.

The present result showed that treatment with 800 mg/kg b.w. of A. inculta repeated after 7, 8 and 9 weeks post infection, resulted in reduction in worms being 46.8%

When treatment was given at a dose of 800 mg/kg b.w. of A. absinthium or A. inculta 7, 15 and 21 days post infection this revealed a highly significant percentage reduction in the mean number of worms (76.3% and 66.3%, respectively).

While the group given a dose of 800 mg/kg b.w. of A. absinthium or A. inculta 24 hours pre- infection and followed  7 and 15 days post infection, the percentage reduction in the mean number of worms was 82.9% and 84.2%, respectively.

Treatment with the same previous doses of A. absinthium or A. inculta 24 hours followed by 7 and 15 days post infection, showed that there was a highly significant percentage reduction in the mean number of worms (77.32% & 74.9%, respectively).

In this work, PZQ, which was given in a dose of 500 mg/kg b.w for two successive days 7 weeks post infection showed that the reduction in worms was 96.4%.

The oogram pattern in the present work showed that, the percentage of S. mansoni immature ova in infected mice were reduced in all groups using the different doses and different time intervals using either plant. There was an increase in dead ova in all treated groups.

The efficacy of PZQ treatment (7 weeks post infection) was shown by the disappearance of the immature stage of the schistosome ova. Moreover, there was an increase in the dead eggs reaching 91.6%.

Treatment with A. absinthium or A. inculta for different time periods showed significant reduction in hepatic and intestinal tissue egg load. This reduction was less marked when both plants were administered 7 weeks post infection or in the group treated 7, 8 and 9 weeks post infection.

Treatment with A. absinthium or A. inculta for different intervals showed statistically significant reduction in egg excretion in stool for all experimental group, while treatment with PZQ showed disappearance of egg excretion in stool.

Administration of A. absinthium or A. inculta led to reduction in hepatic granuloma diameter. This reduction was more marked when either plant was administered 24 hours pre-infection and followed 7 and 15 days post infection (44.55% and 40.37%, respectively).

The number of granuloma showed no significant differences in the different groups, but there was some decrease when the plants were administered 24 hours either pre or post infection and followed 7 and 15 days post infection being 9-11 compared to control group (13-15). There was no improvement noticed in liver pathology.

The highest reduction in granuloma diameter was shown in the group treated with PZQ (57.84%) where the granulomas were lobular and fibrocellular. There was also little decrease in the number of granuloma.

CONCLUSION

In conclusion, our findings revealed that administration of Artemisia plants namely A. absinthium and A. inculta as molluscicidal and schistosomicidal agents resulted in:

1-            A. absinthium ethanol extract proved to have the superior molluscicidal activity against B. alexandrina snails than A. inculta.

2-            S. mansoni cercariae was significantly more susceptible than S. mansoni miracidia to the different concentrations used of either plant extracts helping in the control of snails.

3-            Diminished other aspects of disease morbidity as shown by enhanced reduction in tissue worm load and egg load in tissue and stool with changes in oogram pattern.

4-            Using the ethanol extracts of Artemisia plant showed high efficacy when given in triple doses in early stages of infection than single dose in late stages and when the dose is repeated at this stage.

5-            Decrease in granuloma diameter with no improvement of liver pathology.

 

Recommendations:

The overall aim of the work is to introduce natural compounds as control agents for schistosomiasis to avoid ecologic disturbance together with reducing cost, accordingly:

1-            Using Artemisia plants as molluscicidal agents against B. alexandrina in approach to reduce effects of other chemical molluscicides.

2-            Using repeating doses of Artemesia plants in the early stages of infection with schistosomiasis diseases, can help resolving the practical problem of PZQ increase resistance of schistosomiasis.

3-            Using the Artemesia plants extracts was less costly and more safe than other anti-bilharzial drugs with little (no) side effects. Thus further studies are recommended to extract the effective principle of this plant aiming at better results, especially when liver pathology is considered."