Isolation and immunomodulatory activity of phytoconstituents of Lagenaria siceraria fruits

Medicinal plants are being used since antiquity to treat variety of diseases. Several medicinal plants have been reported to have effect on immune system. The prime objective of ayurveda, one of the ancient Indian system of medicines, is the prevention of disease process. The different health care measures to be adopted by an individual are grouped together under the heading of rasayana. The word rasayana in Sanskrit literally implies the circulation of rasa, the nutrient. Rasayana is antiaging, increases the life span, promotes intelligence and memory and increases resistance to diseases. Rasayana plants possess various pharmacological properties e.g. immunostimulant, antibacterial, antiviral, antiinflammatory, anticancer, antirheumatics etc. Lagenaria siceraria (Molina) Standley is a medicinal plant and utilizable species. The fruits of this plant have been scientifically investigated in experimental animals for their analgesic, anti-inflammatory, antiasthmatic, antihyperlipidemic, antioxidant, antibacterial, diuretic, antidiabetic and hepatoprotective activities. Earlier researchers have reported several phytoconstituents in fruits viz saponins, flavonoids, terpenoids, tannins, carbohydrates etc. However there is paucity of data available on the effect of extracts of fruits of Lagenaria siceraria on immune system. Therefore, the present study was undertaken to investigate the immunomodulatory effect of different extracts/fractions of Lagenaria siceraria fruits in experimental animals. Further from bioactive fractions phytoconstituents were isolated using column chromatography and few of the isolates were again tested for their possible immunomodulatory activity. In present study, dried fruits of Lagenaria siceraria were extracted with methanol using soxhlet apparatus. The concentrated extract was then evaluated for its possible immunomodulatory activity in rats using different immunomodulatory protocols such as haemagglutination antibody titre, carbon clearance and cyclophosphamide-induced myelosuppression. The extract was found to increase antibody titre significantly. It also enhanced rate of carbon clearance from animals‟ blood indicating increased phagocytosis. A significant increase in white blood cell count was observed in methanol extract-treated rats as compared to cyclophosphamide treatment alone. After confirming the preliminary immunomodulatory activity, another batch of dried powder was successively extracted with petroleum ether (60-80), dichloromethane, chloroform and methanol. Methanol extract was then suspended in water and fractionated with ethyl acetate and then n butanol in a separating funnel to enrich successive methanol extract into flavonoids and saponins respectively. Petroleum ether extract, dichloromethane extract, ethyl acetate soluble fraction and n butanol soluble fraction were then investigated for their immunomodulatory activity in experimental animals using following animal models: haemagglutination antibody titre, delayed type hypersensitivity reaction and haematological parameters. Out of them only ethyl acetate and n butanol soluble parts of successive methanol extract were found to be effective. They showed promising immunomodulatory activity. Bioactive fractions were then tested chemically to know the presence of different chemical constituents. TLC and HPTLC studies were also performed to know the number of constituents present in both fractions. Both fractions were then applied separately to glass column chromatography on silica gel (60-120 mesh) to separate possible phytoconstituents by variety of mobile phases. Total five components were isolated in significant quantities and their characterization was done using different spectral techniques (H-NMR, C-NMR, IR and UV analyses) to reveal their identity as oleanolic acid (I), β sitosterol (II), campesterol (III), isoquercitrin (VI) and kaempferol (VII). Two of the isolates namely I and VI were tested pharmacologically to assess their effect on immune system using animal protocols viz haemagglutination antibody titre, delayed type hypersensitivity reaction and carbon clearance parameters. Isolates displayed significant immunomodulator activity. The present study was planned to isolate phytoconstituents from Lagenaria siceraria fruits with minimum side effects and toxicity as compare to synthetic drugs that may offer immunostimulant or immunosuppressant action. Both these effects have significant role in the management of various diseases e. g. immunostimulants will be helpful in the prophylaxis and treatment of some immuno-compromised diseases such as AIDS etc. and against infectious diseases, whereas immunosuppressants would show potential in combating autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis as well as hypersensitivity, graft rejection etc. The identified constituents may serve as lead compound for synthetic procedures. To the best of our knowledge, for the first time, we reported the immunomodulatory property of the fruits of Lagenaria siceraria. The present study with Lagenaria siceraria fruits leads us to conclude that crude methanol extract augments humoral immune response, activates macrophage-induced phagocytosis and prevents cyclophosphamide-induced myelosuppression. Our studies show that Lagenaria siceraria fruits contain flavonoids (isoquercitrin, kaempferol), β sitosterol, campesterol and oleanolic acid; which may account at least in part for their application in folk medicine. This is the first report on the presence of isoquercitrin, oleanolic acid and kaempeferol in the fruits of Lagenaria siceraria. The present study leads us further to conclude that oleanolic acid and isoquercitrin augment humoral immune response, suppress cellular immune response and activate macrophage-induced phagocytosis. Our findings are in agreement with the previously published reports of pharmacological activities of fruits of Lagenaria siceraria, involving immunological background, as also mentioned by many other researchers like antiinflammatory, antibacterial, antioxidant and antihepatotoxic. The results obtained in the present study shown that these extract, fractions and phytoconstituents display significant immunomodulatory effects. Besides from the obvious therapeutic importance, these components would be useful in understanding the mechanism of diseases with higher levels of cellular activation, such as autoimmune and hypersensitivity reactions. These components could serve as lead molecules for development of potential immunomodulators. Further detailed studies are required to elucidate the exact mechanism responsible for immunomodulatory activity of Lagenaria siceraria fruits. The present findings are significant for the development of alternative, inexpensive and perhaps safer strategies for the treatment of diseases requiring use of immunomodulatory agents. Further work on the chemical isolation and structure determination of the compounds from bioactive fractions in order to find the structure activity relationship is warranted. Since secondary metabolite content may vary as a function of multiple factors, such as environmental conditions and harvest period, reproduction of this analysis over a long period of time is needed before the effectiveness of our method is totally demonstrated. Although no immunomodulatory drugs to date have demonstrated overwhelming clinical efficacy in the absence of toxicity, our increasing knowledge of immunopharmacology is cause for great optimism that future agents will provide an important addition to the physician‟s armamentarium.