Study On The Antineoplastic Compounds Of Qinglongyi And Its Mechanisms

Malignant tumors are serious diseases endangering human life and health. The incidence and mortality rate of malignant tumors occupy the first place in many countries and districts around the world. According to the predication of WHO, cancer rates could further increase by 50% to 15 million new cases in the year 2020 and malignant tumor will be the first killer to human health in the 21st Century. Besides, every year there occur about 1.8 million new cancer sufferers and about 1.2 million patients died from tumors in our country alone over the past 20 years. The incidence of cancer has been in a tendency of increase. Malignant tumor is a kind of common and frequent disease, and the pathogeny, the pathology, the clinical manifestations and treatment are complicated. Therefore, it is still a hard work to overcome cancer for medical and health workers globally. As antitumor agent is an important means for tumor therapy, many agents were researched and developed in recent years all over the world. But, most of the agents have many disadvantages, such as expensiveness, many side effects and multidrug resistance. Traditional Chinese medication (TCM) has something unique to Cure Cancer. Screening anticancer constituents and lead compound from TCM has become a research hotpot in the field. According to incompletion statistics, the anticancer agents originated from plant medicine account for 32.25% of all anticancer agents. Paclitaxel, camptothecin and vincristine have been used as principal drug for many kinds of tumors. As the rich resource of the medical plants of our country, it has very important meanings for anticancer agent development and tumor treatment to isolate the anticancer constituents from TCM and confirm their antitumor mechanism.Qinglongyi is the exocarp of immature green fruit of Jugland mandshurica Maxim. and Juglans regia L. It is proved that Qinglongyi has extensive pharmacological effects, such as anti-inflammatory, analgesia, bacteriostasis and anti-tumor. And its noticeable antitumor activity has been widely identified. Up to now, the study on chemical constituents of Qinglongyi is a few, and both the experimental research and clinical application still rest on the stage of crude extraction. The anticancer constituents of Qinglongyi are undefined, which restricts the pharmacological study and further clinical application. To investigate the antitumor material basis and its mechanism, the paper studies mainly from the three aspects. Firstly, the antitumor fractions of the ethanol extract of Qinglongyi were screened by MTT assay in vitro. The most effective fraction, namely the acetic ether fractions, was isolated systematically. The most active constituent, namely juglone, was screened to by MTT assay. Then the extraction, isolation and purification methods of juglone were established. Secondly, the antitumor effect of juglone was confirmed both in vivo and in vitro. Furthermore, the induction of apoptosis and cell cycle arrest by juglone were studied in vitro. Finally, the mechanisms of apoptosis and cell cycle arrest induced by juglone were investigated to discover the exact targets of juglone. The study will offer important theoretical guidance and experimental basis for the new anticancer agent development and clinical application of naphthoquinone.1 Screening of the antitumor compounds in Qinglongyi1.1 Study on the antitumor activity of 4 fractions in different solvents of QinglongyiOBJECTIVE:To estimate the antitumor activity of different solvent fractions of alcohol extract of Qinglongyi and screen the antitumor fractions with MTT assay in vitro. METHODS:Qinglongyi was extracted with ultrasonic extraction by alcohol, and the herb residue was extracted continuously with heat reflux extraction by alcohol. The extracts were collected together, concentrated, removed ethanol completely, and then dissolved in water. The aqueous solution was extracted with petroleum ether, chloroform, acetic ether and n-butanol and each extract was condensed to dry. The antitumor activity of the fractions was evaluated with MTT assay on four kinds of human tumor cell lines (human gastric cancer cell line SGC-7901, human liver cancer cell line HepG2, human lung cancer cell line A549, human breast cancer cell line MCF7) in vitro. RESULTS:The results indicated that petroleum ether fraction, chloroform fraction and acetic ether fraction had inhibitory effect on different tumor cell lines. The IC50 of petroleum ether fraction against SGC-7901, HepG2, A549 and MCF7 was (70.43±2.51)μg/mL, (97.05±2.21)μg/mL, (29.42±1.77)μg/mL and (198.72±12.37)μg/mL respectively. The IC50 of chloroform fraction against SGC-7901, HepG2, A549 and MCF7 was (58.35±1.83)μg/mL, (45.31±1.25)μg/mL, (24.86±1.67)μg/mL and (59.19±3.86)μg/mL respectively. The IC50 of acetic ether fraction against SGC-7901, HepG2, A549 and MCF7 was (51.86±1.16)μg/mL, (28.09±0.69)μg/mL, (25.82±1.51)μg/mL and (47.84±0.80)μg/mL, respectively. The IC50 of n-Butanol fraction against the four kinds of cell lines were greater than 100μg/mL and the water-soluble part had no activity on these cell lines. CONCLUSION:Petroleum ether fraction, chloroform fraction and acetic ether fraction are the antitumor fraction s of Qinglongyi. And the most acitvie fraction is acetic ether fraction.1.2 Studies on the chemical constituents of acetic ether fraction in Qinglongyi OBJECTIVE:To investigate the chemical constituents of acetic ether fraction in Qinglongyi. METHODS:Column chromatography was used to separate and purify the chemical constituents. The structures were elucidated on the basis of physicochemical properties, spectral data and the literature data. RESULTS:Six compounds were isolated and identified as juglone (1),β-sitosterol (2), oleanolic acid (3), vanillin (4), vanillic acid (5) and quercetin (6). CONCLUSION:6 compounds were isolated and were known compounds.1.3 Screening antitumor constituents of acetic ether extract in Qinglongyi OBJECTIVE:To evaluate the antitumor activity of the 6 compounds isolated from acetic ether extract in Qinglongyi. METHODS:The 6 compounds were screened in vitro by MTT assay. RESULTS:Juglone had very strong growth inhibition on four kinds of tumor cells (SGC-7901, HepG-2, A-549, MCF-7) and IC50 was (21.20±0.56)μmol/L, (34.70±0.91)μmol/L, (24.80±0.65)μmol/L and (25.47±0.67)μmol/L respectively. Quercetin had better antitumor activity and IC50 was (53.61±1.41)μmol/L, (47.81±1.26)μmol/L, (57.16±1.51)μmol/L, (54.56±1.44)μmol/L respectively. Oleanolic acid and vanillic acid had antitumor activity on one or two kinds of tumor cells and the antitumor activity needs further investigation.β-sitosterol and vanillin had no antitumor effect. CONCLUSION:Juglone showed significant antitumor activity in vitro. And the antitumor effect and mechanism of juglone worth further studying.1.4 The extraction and isolation of juglone in Qinglongyi OBJECTIVE:To study the method of extraction, isolation and purification of juglone from Qinglongyi. METHODS:The storage years, harvesting periods, drying methods of Qinglongyi and optimize the organic solvent and extraction method of juglone were investigated by HPLC taking the content of juglone as index. The target compound was isolated by silica gel column chromatography and purified by a pre-HPLC. The purity of juglone was determined by HPLC. RESULTS:The suitable harvesting period of Qinglongyi was July to August when the fruits were immature. Qinglongyi should be dried in the shade or in the sun. Storage time of Qinglongyi should not be too long. We should to use the current year medicinal materials as much as possible. Ethyl acetate was the optimum extraction solvent and the optimum extraction method was ultrasound and cold-soaked. The isolation and preparation procedures of juglone as follows:Ethy acetate extract of Qinglongyi was isolated on silica gel column by a gradient elution using CHCl3 and MeOH, tracked by TLC. The same components containing naphthoquinones were complicated. The fraction was isolated continuously on silica gel column by a gradient elution using PE-EtOAC (200:1) elution. The crude juglone was obtained and then was isolated and prepared by semi-preparative HPLC. The purity of refined juglone was 98.23% and the transfer rate was 10.41%. CONCLUSION:The extraction and isolation method established according to the physicochemical properties of jugone is simple, repeatable and more extraction rate. It can be used in the laboratory and industrial production.2 Study on the antitumor effect of juglone in vivo and in vitro2.1 Study on the antitumor effect of juglone in vivoOBJECTIVE:To investigate the antitumor effect of juglone in vivo. METHODS:S180 tumor-bearing mice were set up to investigate the effects of juglone on tumor growth; HE staining was used to observe the growth and pathological changes of tumor cells; the ultrastructural changes of tumor cells were observed by transmission electron microscopy; flow cytometry was used to observe the apoptosis rate of S180 cells; H22 tumor-bearing mice were set up to investigate the effects on survival time. RESULTS:Juglone had growth inhibition effect on S180 solid tumor. The tumor inhibition rate at the dosage of 8μmol/kg of juglone was (48.97±3.27)%. The tumor weight of all dose groups of juglone were lower significantly than that in control group (P＜0.01). Under the optical microscope, the necrosis degree of the tumor cells increased significantly as the concentration of juglone increased compared with the control group. Typical morphological changes of apoptosis were observed under transmission electron microscopy, such as chromatin condensation, margination against nuclear envelope and formation of apoptotic bodies. The result of FCM analysis showed that the subdiploid peak was found and the apoptosis rate increased in a dose-dependent manner. The apoptosis rate of 8μmol/kg of juglone was the highest, reached (10.27±1.05)%. Juglone could increase life span of H22 mice. And the increase in life span of each dose groups was more than 75%. CONCLUSION:Juglone has preferable antitumor effects in vivo and it can induce apoptosis of S180 cells.2.2 Study on the effect of juglone on the inducing apoptosis and cell cycle arrest in SGC-7901 cellsOBJECTIVE:To investigate apoptosis and cell cycle arrest effects of juglone. METHODS: SRB assay was used to investigate the action of growth inhibiting of juglone on SGC-7901 cells; fluorescence microscope was used to observe the apoptotic morphology of SGC-7901 cells; TEM was used to observe the ultrastructure changes of SGC-7901 cells; Cells were doubly marked by Annexin V-FITC/PI, the typical morphological changes of early apoptosis in SGC-7901 cells were observed with LCSM, and the apoptosis rate of SGC-7901 cells was detected; Cells were marked by PI and FCM was used to analysis the cell cycle distribution of SGC-7901 cells. RESULTS:Juglone was found to significantly inhibit cell growth in a dose-dependent manner. GI50 and TGI of juglone (72h) examined by SRB assay were (21.26±0.26)μmol/L and (78.11±3.55)μmol/L, respectively. After exposure to juglone for 72h, thick granulated fluorescence in condensed nuclei and cytoplasm was showed under fluorescence microscope, and the number of apoptotic bodies increased with increasing of juglone concentration. After exposure to juglone for 72h, cells showed typical apoptotic morphology, including decrease in microvilli, crescent margination of chromatin against the nuclear envelope and formation of apoptotic body under TEM. After exposure to juglone for 48h, LCSM analysis showed that SGC-7901 cells appeared modality of early apoptosis at low dose, and the number of early and late apoptotic cells increased in a dose-dependent manner. FCM analysis result showed that the apoptosis rate increased with dose dependent. The apoptosis rate of SGC-7901 cells treated with 20μmol/L of juglone for 48h was (27.60±0.70)%. Cell cycle analysis suggested that after juglone with different concentration (5,10,15,20μmol/L) treated on SGC-7901 cells for 48h, the proportion of G1 phase cell decreased while the proportion of S phase cell increased. It proved that juglone can arrest SGC-7901 cells on S phase. CONCLUSION:Juglone can significantly inhibit the proliferation and induce apoptosis and S phase arrest in SGC-7901 cells.3 Study on the mechanism of apoptosis and S phase arrest induced by juglone on the SGC-7901cells 3.1 Study on the mechanism of apoptosis induced by juglone on the SGC-7901 cellsOBJECTIVE:To investigate the mechanism of apoptosis induced by juglone on the SGC-7901 cells. METHODS:FCM was used to detect the ROS level in SGC-7901 cells; after double stained with Fluo3-AM, the changes of [Ca2+]iin SGC-7901 cells was observed by LCSM; Western blot assay was used to investigate the protein expression of Bcl-2 and Bax; FCM was used to detect the mitochondrial transmembrane potential (△ΨFm) and the protein expression of cytochrome C of SGC-7901 cells; Caspase Assay Kits was used to detect the activity of Caspase-3 in SGC-7901 cells. RESULTS:The results showed that the level of ROS at the dose of 5μmol/L, 10μmol/L,15μmol/L and 20μmol/L of juglone was (34.83±1.45)%, (42.43±1.36)%, (53.73±1.38)% and (68.67±1.33)%, respectively, which were significantly higher than that in control group (P＜0.01). The concentration of Ca2+ in. SGC-7901 cells exposed to juglone for 48h was increased significantly in a dose dependent manner compared with control group(P<0.01). Western blot analysis showed that juglone could decrease the expression of Bcl-2 protein and there was a negative correlation between the protein expression level and the concentration of juglone. Meanwhile, the expression of Bax protein was increased by juglone and there was a positive correlation between the protein expression level and the concentration of juglone. Juglone could decrease the mitochondrial transmembrane potential and the relative mitochondrial transmembrane potential at the dose of 5,10,15 and 20μmol/L of juglone was (85.53±1.82)%, (53.57q2.48)%, (46.33q1.46)%, (36.43q2.64)%, respectively, which were significantly lower than that in control group (P＜0.01). FCM analysis showed that juglone could increase the expression of Cytochrome C protein and there was a positive correlation between the protein expression level and the concentration of juglone. After cells were treated with juglone (5,10,15 and 20μmol/L) for 48h, the relative activity of Caspase-3 increased gradually with increasing juglone concentration, and the active uint was 5.44±0.48,5.65±0.79,7.00±0.83 and 10.44±0.63 respectively. CONCLUSION:Juglone can induce apoptosis of SGC-7901 cells via mitochondria pathway. On the one hand, juglone can down-regulate the expression of Bcl-2 protein and up-regulate the expression of Bax protein, which promote MPTP opening and in turn decrease the mitochondria membrane potential of SGC-7901 cells. On the other hand, juglone can increase intracellular level of ROS, which increase the intracellular concentration of Ca+ and lead to mitochondrial Ca2+ overload, thus inducing the opening of MPTP. Meanwhile, the high level of ROS can also cause MPTP opening directly and decrease the membrane potential of mitochondria in SGC-7901 cells.The decrease of mitochondria membrane potential can conversely promote the production of more ROS, which lead to more serious injury of mitochondria and release of Ca2+ and other contents from mitochondria to the cytoplasm. At the meantime, the decrease of mitochondria membrane potential can also cause relative high permeability in mitochondria membrane, substrate expansion of mitochondria, rupture of outer membrane and release of Cytochrome C. The release of Cytochrome C in Cytoplasm can activate Caspase cascades reaction, finally initiate the activating of Caspase-3 and induce cell apoptosis.3.2 Study on the mechanism of S phase arrest induced by juglone on the SGC-7901 cellsOBJECTIVE:To investigate the mechanism of S phase arrest induced by juglone on the SGC-7901 cells. METHODS:Western blot assay was used to investigate the S phase correlative protein expression of CHK2, CDK2, Cdc25A and p-Cdc25A. RESULTS:Western Blot result showed that juglone could upregulate the CHK2 protein expression and downregulate the expression of CDK2 protein with dose-dependen. There was negative correlation between Cdc25A protein expression level with juglone dose, but positive correlation between p-Cdc25A protein expression level with juglone dose. CONCLUSION: The mechanism that juglone induced S phase arrest of SGC-7901 cell might be that juglone induces cells DNA image and actives CHK2 protein which can phosphorylates and inactivates downstream target protein Cdc25A. The inactivated Cdc25A cannot dephosphorylate Tyr15 of CDK2 and in turn down-regulates the activity of CDK2. It makes CDK2 lose the effect of starting and promoting S phase progress which leads to S phase arrest of SGC-7901 cell.CONCLUSIONPetroleum ether fraction, chloroform fraction and acetic ether fraction are the antitumor active fractions of Qinglongyi and the acetic ether fraction is the most active fraction.6 compounds are isolated and identified from acetic ether fraction of Qinglongyi. They are juglone,β-sitosterol, oleanolic acid, vanillin, vanillic acid and quercetin. All the 6 compounds are known compounds, and quercetin and especially juglone have outstanding antitumor activity in vitro. Human gastric cancer SGC-7901 cell line is the most sensitive cell line to juglone.By using ethyl acetate as extraction solvent, ultrasound and cold-soaked methods as extraction methods, silica gel column chromatography as isolation method, we get crude product of juglone. Then it is purified by semi-preparative HPLC. The purity of refined juglone was 98.23% and the transfer rate was 10.41%. The requirement of riginal medicinal of Qinglongyi:Storage time of Qinglongyi should not be too long. We should to use the current year medicinal materials as much as possible; the suitable harvesting period of Qinglongyi was July to August when the fruits are immature; Qinglongyi should be dried in the shade or in the sun.Juglone has favorable antitumor effects both in vivo and in vitro. The mechanism might be that juglone can induce apoptosis and cell cycle arrest. On the one hand, juglone can induce cancer cell apoptosis via mitochondria pathway by increasing the intracellular level of ROS and influencing the protein expression of Bcl-2 and Bax. On the other hand, juglone induces cells DNA damage and leads to S phase arrest of cancer cells via ATM-CHK2-Cdc25A-CDK2 regulation approach.INNOVATIVE POINTS OF CURRENT STUDY1. By using bioactivity-guided targeted screening in vitro the antitumor fractions and constituents of Qinglongyi are confirmed.2. Extraction, isolation and purification technology of juglone, an unstable naphthoquinones composition, is established. The purity and extraction rate of juglone are higher than that has been reported ever before.3. It illustrates that juglone can induce apoptosis in human gastric cancer SGC-7901 cells via the mitochondrial pathway.4. It illustrates that juglone can induce S phase arrest via ATM-CHK2-Cdc25A-CDK2 regulation approach.