| Longan (Dimocarpus longan Lour.), which originated from Southeast Asia, is a non-climacteric subtropical fruit of the Sapindaceae family. Longan fruit has a thin, leathery and indehiscent pericarp surrounding a succulent edible aril with a large dark brown seed. Longan fruit is rich in carbohydrates, protein, fiber, fat, vitamin C, amino acids, and minerals and so on. It has been used in traditional Chinese medicine for tonifying both the heart and spleen, and serving as an agent in relief of neural pain and swelling. In recent years, it has been found that Longan has some pharmacological functions such as antitumor, antioxidation, and senescence delay and immunity enhancement. Herein, the study aims at studying chemical constituents and the pharmacological activities of longan and lays the foundation for preliminarily clarifying the relation between its chemical constituents and efficacy to unearth its effective constituents and develop new medicine.1ObjectiveAfter extraction and preliminarily separation of longan chemical constituents, screening of its active components, and then systematic study on its chemical constituents, substance categorization and structure of main active components have been confirmed. The same time, its antitumor effect and general pharmacology are studied. All these studies will offer experimental data for further development and utilization of longan. 2Method2.1Extraction, Separation and Identification of the Chemical Constituents from Different Parts of Longan.With95%ethanol as extraction solvent, the main chemical constituents of longan aril, longan husk and longan kernel were extracted by reflux extraction. The physical and chemical characteristics were preliminarily identified with some types of chemical reagents. Extracting three parts of longan with petroleum ether, ethyl acetate, n-butyl alcohol respectively, different polar extracts from the three parts of longan have been obtained, and then weighed and calculated its productivity.2.2Screening Different Parts of the Longan Aril for Its Antitumor EffectsThe model of mice transplanted sarcoma S180is used to screen different parts of longan aril for its anti-tumor effects.2.3Study on Chemical Constituents of Logan Active ComponentsWith the result of antitumor effects of longan aril, the acidity and physicochemical properties of the yellow brown crystalline compounds from longan aril are determined and identified, and then scanned by UV and detected by HPLC to define its constituent types. The main components of longan flavonoid glycosides are studied further by extracting and purifying with thin layer chromatography, silica gel column chromatography, polyamide column chromatography, sephadex gel chromatography, macroporous resin column chromatography and high performance liquid chromatography etc. The purity and structure of the separated and purified chemical compounds are preliminarily identified by melting point, HPLC, UV and NMR etc. In addition, petroleum ether extract from three parts of longan have been analyzed by GC-MS.2.4Study on Preliminary Pharmacological Effects of the Longan Flavonoid Glycosides2.4.1Study on Antitumor Effects in Vitro:Effects of longan flavonoid glycosides on the proliferation of four human carcinoma cells, HepG2, HeLa, A549and U251cultivated in vitro were measured by MTT.2.4.2Study on Antitumor Effects in Vivo: The model of mice transplanted sarcoma S180is used to observe and detect the influence of tumor inhibition made by intragastric administration of longan flavonoid glycosides.2.4.3Study on General Pharmacological Activities:The effects of longan flavonoid glycosides on mice nervous system were measured by using field experiment, sleep and tail suspension test. The effects on breathing rates of anesthetic mice and blood pressure of anesthetic rats treated with longan flavonoid glycosides were measured through recording and analyzing physiology with RM-6280. Furthermore, the influence on the respiratory system and cardiovascular system has been analyzed.3Results3.1Extraction, Separation and Identification of Chemical Constituents of Different Parts of Longan.3.1.1Extraction and Separation of Chemical Composition of Different Parts of Longan95%ethanol extract of Longan aril, husk, kernel were extracted by three different polar solvents of n-butyl alcohol, ethyl acetate, petroleum ether. As a result,14.5g petroleum ether extract,36.3g ethyl acetate extract and118.0g n-butanol extract from5kg longan aril has been obtained respectively, and their corresponding productivities were0.3%,0.7%,2.4%;31.1g petroleum ether extract,47.5g ethyl acetate extract,74.2g n-butanol extract have been obtained from3kg longan husk respectively, and their corresponding productivities were1.0%,1.6%,2.5%;114.8g petroleum ether extract,78.1g ethyl acetate extract,89.8g n-butanol extract have been obtained from5kg longan kernel respectively, and their corresponding yield were2.3%,1.6%,1.8%.70.1g yellow brown crystalline substances have been obtained from longan aril, the yield was1.4%.3.1.2Physical and Chemical Identification of Different Chemical Constituents of Logan FruitThe physical and chemical identification experiment on the95%alcohol extraction of longan aril, husk and kernel showed that2%aluminium trichloride, iso-hydroxyl oxime acid iron reaction reagent, acetic anhydride-concentrated sulfuric acid reactions of alcohol extracts from three parts were all positive;10%potassium hydroxide, bismuth potassium iodide reactions were all negative;1%ferric chloride and ninhydrin reactions of extract from longan husk and kernel were positive, but that from longan aril was negative; bromophenol blue reagent reactions of extract from longan aril and husk were positive, but that from longan kernel was negative.3.2Screening Different Parts of the Longan Aril for Its Antitumor EffectsThe results of antitumor activity in vivo showed that the different parts of longan aril all displayed anti-tumor in different degree, The tumor inhibition rates of95%alcohol extract, yellow brown crystalline substances, petroleum ether parts, ethyl acetate parts and n-butanol parts from longan aril were26.2%、41.3%、28.9%、14.3%、34.3%respectively.3.3Study on Chemical Constitutes of Longan Active Components3.3.1Separation and Analytical the Structure of Chemical Constituents of Logan Active SitesThe result of acidity test showed that the pH of the yellow brown crystalline compounds from longan aril was about three, the physicochemical identification showed that the a-naphthol sulfuric acid, hydrochloric acid-magnesium powder, phosphomolybdic acid reagent, aluminum salt and magnesium salt complexation reaction, ammonia fumigation reactions were all positive. Under ultraviolet light, there was yellow-green fluorescence. With UV scanning the maximum absorption of longan crystalline compounds was at295nm. The purity of componds A and B separated from longan active sites by the HPLC were90%and92.0%respectively. The ultraviolet scanning showed that the maximum absorption of compounds A and B were at297.8nm and284.7nm respectively. The meting range of compound A was132℃-135℃, and that of compound B was161℃-163℃. The maximum absorption wavelengths were297.8nm and284.7nm respectively by UV scanning.The result of NMR scanning of compound A showed that13C and1H signal peaks of part of flavonoid glycosides.3.3.2GC-MS Analysis on Longan Fruit of Different Parts The compositions of petroleum ether extracted from of longan aril, husk, and kernel had been analyzed by GC-MS.25compositions were separated from longan aril and15compositions were identified. Its identification rate was60.0%, and identified substance accounted for76.7%of the total petroleum ether. The main content of compositions were squalene, hexadecanoic acid, di-isobutyl phthalate, oleic acid, ethyl oleate and ethyl palmitate.45compositions were separated from longan husk and28compositions were identified. Its identification rate was62.2%and its identified substance accounted for83.2%of the total petroleum ether. The main content of compositions were gammn-sitosterol, hexadecanoic acid, fridelan-3-one,9,12-octadecadienoic acid,(Z)-9-octadec-9-enoic acid, squalene, stigmasterol.43compositions were separated from longan kernel and30compositions were identified. Its identification was69.8%and identified substance accounted for87.1of the total petroleum ether. The main content of compositions were hexadecanoic acid, oleic acidand ethyl oleate.3.4Study on Preliminary Pharmacological Effects of Longan Flavonoid Glycosides3.4.1Antitumor Effects in VitroThe longan flavonoid glycosides could inhibit the growth of the four kinds of tumor cell in different degrees. In the drug concentration of1000mg/L, tumor inhibition rates of longan flavonoid glycosides were:53.2%(HepG2),48.7%(HeLa),79.5%(A549) and41.6%(U251). There was obvious concentration-dependent relationship. When the drug concentration ranged from10mg/L to1000mg/L, the survival rates reduced from98.2%to53.2%, on HeLa cells from96.2%to48.7%, and on U251cells from87.9%to41.6%.3.4.2Antitumor Effect in vivoThe result shows that three doses of the flavonoid glycosides could all inhibite the growth of tumor in mice with a dose-dependent relationship. When the drug concentration increased from60mg/kg to240mg/kg, the tumor inhibition rates increased from13.0%to52.5%. Comparing with control group (2.18mg/g,7.18mg/g), the thymus index (0.68mg/g) and the spleen index (3.72mg/g) of5-fluorouracil group decreased obviously, and the index of the three doses of flavonoid glycosides groups were obvious higher than the5-fluorouracil group. But with the increase of the dose, the thymus index had a tendency to decrease (from2.06mg/g to1.63mg/g); the spleen index has a trend to increase (from8.85mg/g to11.94mg/g).3.4.3The General Pharmacological EffectsThe flavonoid glycosides of longan in middle dose (120mg/kg) and high dose (240mg/kg) have certain inhibition on mice autonomic activities (P<0.05). But the three-does groups had no obvious effect on respiratory rate and its blood pressure.4Conclusions4.1Extraction, Separation and Identification of Chemical Constituents from Different Parts of Longan95%ethanol extraction of longan aril, longan husk and longan kernel were refined with petroleum ether, ethyl acetate, n-butanol extract, respectively. Results showed that compounds with high polarity were rich in longan husks and arils, but fat compounds were rich in longan kernel. The result of physiochemical property identification showed that there were slight differences on the categorizations of compounds in different parts of longan, longan aril, husk and kernel. Flavonoids, organic acids, lactones or coumarins, saccharides, saponins were contained in longan aril, husk and kernel. Furthermore, compounds such as tannins, amino acids or peptides or proteins were contained in longan husk and kernel.4.2Screening Different Parts of the Longan Aril for Its Antitumor EffectsThe results of antitumor activity in vivo showed that all chemical constituents parts of longan aril have certain anti-tumor activity, and the yellow brown crystalline substance had better antitumor effects.4.3Study on Chemical Constituents of Longan Active ComponentsThe results of acidity test, physicochemical identification and UV scanning for yellow brown crystalline compounds from longan aril showed that they were flavonoid glycoside. Because the qualities of compound A and B separated from longan flavonoid glycosides were less, and their purity were, their structures will be analyzed further. GC-MS analysis of the various parts of longan displayed the chemical constituents of petroleum ether fraction, and provided reference for other research.4.4Study on Preliminary Pharmacological Effects of Active Components in Longan Flavonoid GlycosidesThe studies on its in-vivo and in-vitro antitumor effect showed that the active components in longan flavonoid glycosides had anti-tumor and immune regulation effects. General pharmacology studies showed that the components in longan flavonoid glycosides with test does (120mg/kg、240mg/kg) had certain inhibitory effects on mice nervous system, and no obvious influence on respiration rate. However, the group with middle dose had a trend to slow the respiration rate and no significant effect on blood pressure of the rats.According to the above study, it shows that longan flavonoid glycosides may be the main active ingredients of antitumor effect, immunity enhancement and tranquilization and allaying excitement. At present, the technology to extract, separate and purify flavonoid glycosides material from longan aril has been preliminarily established. All these studies will provide reference for further study on chemical constituents of longan and new drug development. |
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