PHYTOCHEMICAL ANALYSIS OF HYPTIS SUAVEOLENS EXTRACT AND ITS EFFECTS ON THE GROWTH OF DISEASE-CAUSING PARASITES

Plants extracts contain many essential natural synthesized chemical compounds with for medicinal and application. There has been a growing concern about the adverse effects of mosquito repellants and the need to search for natural and environment-friendly Mosquito repellants. Synthetic insecticides and their associated toxicity issues and the growing incidence of insect resistance have inspired novel insecticides. The present study analyzed the phytochemical extract of H. Suaveolens its effect on mosquito pupa. reveals

Plants extracts contain many essential natural synthesized chemical compounds with considerable potentials for medicinal exploitation and application. There has been a growing concern about the adverse effects of mosquito repellants and the need to search for natural and environment-friendly Mosquito repellants. Synthetic insecticides and their associated toxicity issues and the growing incidence of insect resistance have inspired novel insecticides. The present study analyzed the phytochemical extract of H. Suaveolens its effect on mosquito pupa. A total number of pupa (320) was poured into a 200ml glass beaker; twenty pupae each per three hours was introduced into a glass beaker containing different extract concentrations (ppm) of Hyptis suaveolens. Twenty pupae were introduced in contrast to a glass beaker containing 100ml of distilled water treated with 1ml of acetone used as treated control. Twenty pupae were introduced into 100ml of distilled water and used as untreated control. The result reveals that Hyptis suaveolens possesses inhibitory activity against Anopheles gambiae  (Santos et al., 2007). However, biological attributes of the plant have been well documented owing to its good medicinal value due to the presence of essential oils, alkaloids, flavonoids, phenols, saponins, terpenes, and sterols. In traditional medicine, the plant leaves are applied as insectifuge because of their intense aroma, especially against mosquitoes.

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Mosquitoes are a significant global public health concern, with a concomitant increase in people at risk of infection (Guégan et al., 2018). It has become the oldest human enemy and represents a significant threat to human health because of its ability to vector pathogens that cause diseases like Dengue fever, Dengue hemorrhagic fever, Malaria, Japanese encephalitis, and Filariasis that afflict millions of people worldwide. ( There has been a growing concern about the adverse effects of Mosquito repellants. There has been an increasing need to search for natural and environment-friendly mosquito repellants (Singh et al., 2011). Synthetic insecticides and their associated toxicity issues and the growing incidence of insect resistance have inspired novel insecticides. Plant extracts may be alternatives sources that constitute a rich source of bioactive compounds that are biodegradable and environmentally friendly. Research has shown that H. suaveolens an invasive weed with insecticidal properties (Benelli et al., 2012;Devi Priya, 2016;Sharma et al., 2019). Thus, the current study intends to explore the inhibitory role of H. suaveolens aqueous extract on the pupa of mosquitoes.

Collection of Anopheles mosquito pupa
The anopheles' mosquito pupa was collected from stagnant waters at various locations in Kogi state using dipper and pipette as described. The collected pupa was free from tadpoles which may cause its mortality. The pupa was reared in a clean white plastic bowl covered with a fine and clean mosquito net.

Plant source
The plant Hyptis suaveolens leaves were collected and confirmed by a Botanist. The leaves were washed with distilled water and sliced into small pieces, and dried.

Preparation of Crude Extract Soxhlet Extraction
The crude extraction of Hyptis suaveolens was done using the Soxhlet extraction technique as describe by Zygler et al. (2012). The coarse powder (200g) of Hyptis suaveolens leaves was gained by pounding the air-dried leaves with a mortar and pestle. The coarse powder was transferred into the Soxhlet extractor column and measured into the flat bottom flask and the Soxhlet. A reflux condenser was inserted into the Soxhlet, and rubber hoses connected from the condenser to the circular water. The setup was placed in a heating mantle, and the mantle was connected to the main connection.

Preparation of Test Concentration
Plant crude extract was prepared through a single dilution method by mixing it with distilled water, each in a sterile glass beaker. Treated control was prepared and kept safe before pupa inhibitory bioassay.

Pupa Inhibitory Bioassay
Pupa bioassay will be prepared according to a standard procedure provided by the world health organization, guideline for laboratory and field testing of mosquito pupacide (WHO, 2005). The pupa was transferred through strainers of droppers to labeled sterile glass beakers of concentrations. The pupa inhibitory activities of each extract concentration were evaluated by counting the number of dead pupae for the period. Pupa was confirmed dead when no movement is observed and no response to a stimulus when touched with a pasture pipette. The dead pupa was carefully removed from the setup and placed in a clean filter paper, counted, and recorded at some intervals.

Result: -
Tables containing the number of dead and live pupa against each concentration were made. Degree activities were evaluated by plotting the number of dead pupae against concentration. The percentage of dead and alive for every interval will be determined.  20  0  0  0  50ppm  20  2  5  11  100ppm  20  3  6  15  150ppm  20  5  8  17  200ppm  20  9  11 19    Treated control  20  0  0  0  Untreated control  20  0  0  0  50ppm  20  9  45  55  100ppm  20  10  50  50  150ppm  20  13  65  35  200ppm  20  15 75 25  Treated control  20  0  0  0  Untreated control  20  0  0  0  50ppm  20  11  55  45  100ppm  20  15  75  25  150ppm  20  17  85  15  200ppm  20  19  95  5 Discussion: -Natural pesticides, especially those derived from plants, are more prosing and effective in mosquito (pupa) control (Amer & Mehlhorn, 2006). Hyptis suaveolens were found to have some pupa inhibitory activity against 558 mosquito pupa at different concentration rates in part per million (ppm), as shown in table 1. The result showed the net change in the death rate of pupa with a subsequent increase in its concentration compared to control. A total number of pupa (320) was poured into a 200ml glass beaker; twenty pupae each per three hours was introduced into a glass beaker containing different extract concentrations (ppm) of Hyptis suaveolens. Twenty pupae were introduced in contrast to a glass beaker containing 100ml of distilled water treated with 1ml of acetone used as treated control. Twenty pupae were introduced into 100ml of distilled water and used as untreated control.
Table1 shows the effect of different concentrations of crude extracts of Hyptis suaveolens on Anopheles Gambiae larvae at different time intervals. After 12hours, no motility was recorded for treated and untreated control. In three (3) hours, ±2 pupa was recorded dead against a concentration of 50ppm. After three hours, twenty (20) pupa was introduced into another glass beaker containing 50pm of crude extract and allowed to stand. In six (6) hours, ±5 pupa was recorded dead. The same procedure was repeated for the 9 th and 12 th hours, respectively. In the 9 th hour, ±9 pupa was recorded dead, and in the 12 th hour, ±11 pupa was recorded dead. The same procedure was repeated for concentrations 100ppm, 150ppm, and 200ppm. At concentration 100ppm, in 3hours, 6hours, 9hours and 12hours, ±3, ±6, ±10, and ±15 pupa was recorded dead, respectively. At concentration 150ppm, in 3hours, 6hours, 9hours, and 12hours, ±5, ±8, ±13 and±17 pupa was recorded dead. At concentration 200ppm, in 3hours, 6hours, 9hours and 12hours, ±9, ±11, ±15, and ±19 pupa was recorded dead. Percentage of the dead for pupa that died in 3hours, 6hours, 9hours, and 12hours were calculated by dividing the number of pupae dead after treatment with several controlled (20) and multiplying with 100%, and percentage alive for the remaining pupa was calculated by subtracting percentage dead from 100%. This procedure was used in the following tables. The inhibitory activity was found to be higher against the pupa at the highest concentration rate of 200ppm.

Conclusion: -
Plant extracts in insect/mosquito control are an effective pest control method and help minimize the aggressive growth of mosquitoes. The result reveals that Hyptis suaveolens possesses inhibitory activity against Anopheles gambiae. Therefore, the result contributed to the literature by laying the ground for further analysis of the bioactive constituents of Hyptis suaveolens extract and its systemic effects on target mosquitoes. This may enable the application of the extract as a pupa inhibitor in a considerable area of aquatic habitats or breeding sites in and around human dwellings for effective control of vector mosquitoes.

Funding
Tetfund funds the research