In-Vitro Evaluation of Anti-Urolithiatic and Larvicidal Activity of Alternanthera sessilis

Evaluation of Anti-urolithiatic and Larvicidal activity of Alternanthera sessilis. Method: The whole plant of Alternanthera sessilis were extracted using ethanol as solvent. Then it was evaluated for its phytochemicals and later on in vitro anti-urolithiatic study was conducted on the plant using the methods titrimetry, simultaneous flow static model, turbidimetry and gravimetric. The plant showing larvicidal effect was determined by larvicidal assay method. Result: The ethanolic extract of the plant showed the presence of various phytochemicals like phenols, flavonoids, tannins, sterols, saponins. The anti urolithiatic activity conformed that the plant can effectively mineralise calcium oxalate in a dose dependent manner when compared to control and standard. The plant also possesses larvicidal activity and the percentage mortality exhibited a dose dependent manner. Conclusion: The ethanolic extract of the plant possessed antiurolithiatic as well as larvicidal activity.

Medicine being the science of diagnosing, treating or preventing diseases and damage to the mind or body . A field that goes hand in hand with innovation and in fact being the one that requires remodelling. Today, creating an effective treatment for disease and injuries by building on existing discoveries and knowledge. Medicines along with its new technology have become the antidote for illness and injuries that resulted in their destruction in the entire society. Medicinal plants being used in healthcare system since time immemorial and studies being carried out all over the world in order to verify their efficacy and some of the discoveries have led to the production of plant based medicine. However conscious efforts are always needed to be made to properly find out, recognise and set up medicinal plants in the design and implementation of these strategies. These kind of approach provide interesting and emerging perceptive in the field of medicinal plants. Alternanthera sessilis 1,2,3,12 a widespread herb found throughout the tropics and the subtropics that is either annual or perennial with several spreading branches bearing small petioled leaves with tiny white flowers found mainly ascending to an altitude of 1200 metres in the himalayas, or could be even cultivated as a potherb. This being used in Indian traditional system of medicine in complications due to vitiated blood , skin disease and ulcer its active principle that is usually being extracted in oil were used to treat infected wounds and its nutritive value makes the herb a potent toxic with a wide range of application. Poultice of pounded fresh material is used in sprains burns and eczema whereas a decoction is recommended for wounds, flatulence, nausea, vomiting cough, bronchitis, diarrhea, dysentery and diabetes with its roots being able to relieve inflamed wounds and applied externally on acne and pimples. A sessiles plant has been reported to posses antimicrobial, molluscicidal, moderate anti mutagenic, antidiarrhoeal, hepato protective cytotoxic and antiviral activities. Phytochemical studies that were done reported the isolation of flavonoids, triterpenoids, steroids and tannins. B-sitosterol, stigmasterol, campsterol, luprol being few of the important constituents. The petroleum ether and benzene extract inhibit the growth of some human and plant pathogenic bacteria. Previous studies showed that that they had hepato protective and potent nootropic activity.

Urolithiasis
The formation of stones in the kidney , bladder or urethra . Stones form twice as often in men than in women The stones formed in the pelvic region cause radiating pain to the groin areas. The stone being mainly composed of calcium oxalate(68.8%) , ammonium phosphate(71.2%) and magnesium.
Medical treatment include acute pain management with NSAIDs chronic stone management using thiazide diuretic, potassium sparing diuretic, calcium channel blocker , allopurinol with alkalisation of urine and increase in fluid intake. Phyto,therapeutic agents are alternatives in treating this using different mechanisms such as antimicrobial, anti oxidant diuresis with increased urinary citrate excretion. In vitro and in vivo analyses are performed on medicinal plants for their urolithiatic property. A normal range of uric acid being 250-750 mg per 24 hours in urine, a higher value indicates gout or kidney stones other causes are a diet high in food containing purines.

Larvicidal Activity
In epidemic disease such as malaria, filariasis, dengue etc that are transmitted by mosquitoes vector control is an important aspect. Excessive use of synthetic pesticides causes pesticide resistance and unwanted effects on non targeted organism. These factors have led to an urgent requirement for development of improved mosquito control method that are cheap , feasible as well as safe for non targeted organism and the environment. Herbicidal insecticides become a priority in this matter, some potentially active larvicides of plant origin like falcarinol, geraniol, azadiractin, plumagin, B-sitosterol etc have been isolated so far. In a bioassay of 72 hours with crude plant extracts, the highest mortality was recorded to be 1.5%. A sessiles showed the highest mortality (76.7%) at 1.5 %crude extract against larvae, this activitywas in the targeted species (Culex quinquefascioctus). The biological activity of secondary compounds of the plant serves to have greater importance. Studies show saponins as the active component. Saponins being soluble in organic solvents and water, act by associating with cuticle membrane of larvae leading to destructing the membrane, causing larval death.

Preliminary Phytochemical Screening
The ethanolic extract was treated with various chemical reagents ,for the qualitative analysis of phytoconstituents like alkaloids, glycosides, phenolic components, flavonoids, tannins, saponins, steroids, carbohydrates, terpenoids etc and their respective chemical tests were performed.

In Vitro Anti-Urolithiatic Activity
Evaluation of calcium oxalate by titrimetry 4, 5, 6 simultaneous flow static model 7 , gravimetric assay 8 and turbidimetry 9, 10 was performed. Larvicidal Activity 11 The percentage mortality of mosquito larvae was evaluated against different concentration of ethanolic extract of A.sessilis. The larvae were considered dead if they settled and remained motionless in the bottom of the container with no response to light or mechanical stimulus.

Extraction
Shade dried Alternanthera sessilis whole plant was extracted with ethanol by using soxhlet

Simultaneous Flow Static Model
From the study result, it was observed that the percentage inhibition is directly proportional to the amount of extract taken. The maximum percentage inhibition of 66.45% was shown by 40 mg of the extract and the minimum inhibition i.e., 25.18% is shown by 10 mg of the extract. The percentage inhibition exhibited by standard cystone was 69.40% Gravimetry From the result it was seen that the percentage dissolution was linear to the amount of crude plant extract taken. The maximum dissolution of 54.92% was shown by 100 mg of plant extract and the minimum dissolution of 17.54% exhibited by 10 mg. The standard cystone (10 mg) exhibited 60% dissolution.

Turbidimetry
The effect of plant extract to various phases of calcium oxalate crystallisation was determined by measurement of turbidity. From the above result it can be concluded that the percentage inhibition was directly proportional to the amount of extract present. The maximum percentage inhibition of 69.50% was shown by 100 mg of plant extract and minimum inhibition of 69.50% was shown by 10 mg of the extract. The standard drug cystone showed a percentage inhibition of 82.85%. Plant extracts are usually a combination of several phytoconstituents. The plant Alternantherasessilisis a good source of flavonoids, saponins, tannins, sterols, phenolics, triterpenoids. From the entire test conducted to evaluate the anti-urolithiatic potential, it was observed that the plant extract exhibits a dose dependent inhibition. It was already proved that flavonoids show good anticristallo-oxalocalcic activities. Flavonoids reduce oxalocalcic crystallization. According to the work done be Vennila V et al it was proved that saponins are also known to possess anticrystallisation property by disaggregating the suspension of mucoprotiens which are promoters of crystallization. Another study conducted by Anbu J in 2011 proved that triterpenoids also possess antiurolithiatic property. They act by increasing the urine output thus reducing the risk of calcium oxalate super saturation in urine. The phytoconstituents could be the reason for antiurolithiatic action of the plant. Further study need to be conducted to know which of these phytoconstituents contribute more to the antiurolithiatic effect exhibited by the plant.

Larvicidal Activity
The larvicidal action of the plant shows linearity to its concentration. The 100µg/ml concentration showed the highest percentage mortality which was found to be 75.18%. The lowest percentage, 11.11% was shown by the lowest concentration 20ìg/ml. The LC50 value was calculated and the concentration was found to be 66.84µg/ ml. This work demonstrates the effect of Alternanthera sessilis on the mosquito larvae. The ethanolic extract of the plant seemed to have concentration dependent effect over the larvae. The presence of phytoconstituent saponin in the plant lead to the study, it was on an assumption that the presence of saponin in the plant can affect the mortality of larvae. The larvicidal effect of plants with the presence of saponin was studied earlier by Bishnu Chapagain and Zeev Wiesman 2005. Taking these studies into consideration A.sessilis was evaluated and the plant showed considerable effect when compared with other studies. Saponin molecules interacting with the larval cuticle membrane causes disruption of the membrane, leading to larval death. Further studies have to be conducted to evaluate and confirm whether saponin is responsible for the action and the mechanism involved.

CONCLUSION
In vitro studies that determine the anti urolithiatic activity and larvicidal activity of alternantherous sessilis was performed .Some constituents like saponins , flavanoids ,tannins ,sterols, phenolic compounds were found after the phytochemical evaluation of the plant. The antiurolithiatic activity was analysed using methods like titrimetic, gravimetric and turbidimetric evaluation. The above constituents could be the reason for the activity of the plant. Positive results  were also shown during the preliminary evaluation of larvicidal activity and presence of saponins could be the reason for this. The studies only provide preliminary information of the activity of the plant. Future studies may be conducted to understand the exact mechanism of action of each of these constituents present in the plant.