Anti-hepatitis, antioxidant activities and bioactive compounds of Dracocephalum heterophyllum extracts
© The Author(s) 2016
Received: 22 April 2016
Accepted: 1 July 2016
Published: 6 August 2016
Dracocephalum heterophyllum was a traditional Tibetan medicine possesses various pharmacological effects involved in anti-inflammatory, antibacterial activities. However, its anti-hepatitis, antioxidant activity and bioactive compounds have not been reported, the objective of this research work was to investigate the pharmacological activity and bioactive compounds of D. heterophyllum extracts.
In the present study, the anti-hepatics and antioxidant activities of four D. heterophyllum extracts (i.e. petroleum ether extracts, ethyl acetate extracts, n-BuOH extracts, and water extracts) were conducted. The main chemical constituent of petroleum ether and ethyl acetate extracts were also isolated using chromatographic techniques and identified by NMR spectroscopic methods. The anti-hepatitis assay showed that the petroleum ether and ethyl acetate extracts of D. heterophyllum significantly prolonged the mean survival times and reduced the mortality of mouse hepatitis model induced by concanavalin A (ConA). The levels of alanine transaminase, aspartate transaminase in blood serum could be decreased obviously by ethyl acetate extracts compared with ConA group (P < 0.01). The histological analysis demonstrated that the ethyl acetate extracts could inhibit apoptosis and necrosis caused by ConA. In addition, the antioxidant activities of the four extracts of D. heterophyllum were measured by DPPH assay, ABTS assay, anti-lipidperoxidation assay, ferric reducing antioxidant power assay, ferrous metal ions chelating assay and determination of total phenolic contents. The results showed that the ethyl acetate extract had the highest antioxidant activities, followed by petroleum ether extract. Finally, nine mainly compounds were isolated from the Petroleum ether and ethyl acetate extracts, including four triterpenes: oleanolic acid (1), ursolic acid (2), pomolic acid (3), 2α- hydroxyl ursolic acid (4), three flavonoids: apigenin-7-O-rutinoside (5), luteolin (8), diosmetin (9) and two phenolic acids: rosmarinic acid (6), methyl rosmarinate (7).
The Ethyl acetate extract of D. heterophyllum had the highest anti-hepatitis and antioxidants activities, followed by petroleum ether extract. The bioactive substances may be triterpenes, flavonoids and phenolic acids, the ethyl acetate extracts of D. heterophyllum may be possible candidates in developing anti-hepatitis medicine.
Hepatitis is an inflammation of the liver. The condition can progress to fibrosis (scarring), cirrhosis or liver cancer. Hepatitis viruses are the most common cause of hepatitis in the world. There are five main hepatitis viruses, referred to as types A, B, C, D and E. In particular, types B and C lead to chronic disease in hundreds of millions of people and, together, are the most common cause of liver cirrhosis and cancer. More than 20 million people worldwide are infected with hepatitis virus. And infection from these viruses results in approximately 1.45 million deaths each year. Effective prophylactic vaccines have been available since the 1980s (Hollinger and Liang 2001). Nonetheless, for many developing countries, large-scale vaccination programs were hardly affordable, and an enormous number of chronic hepatitis virus carriers will be in need of better medication for decades to come. Current therapies are based on the systemic administration of high doses of interferon-α (IFN-α) or on nucleoside analogs. However, both therapies have a sustained response rate of only about 30 %, combinations exert no clear synergism, and lamivudine therapy leads to the rapid emergence of resistant virus variants (Pumpens et al. 2002; Zoulim 2001). Hepatitis B virus (HBV) is a hepadnavirus DNA virus with species specificity and tissue specificity, normally infect only humans and chimpanzees. There is no other feasible small experimental animal infection model. It is also very difficult to infect the cells cultured in vitro. For now, though there are many hepatitis animal model, such as duck hepatitis B model (Schultz et al. 2004), woodchuck model (Wang et al. 2004), chimpanzees model (Wieland et al. 2004), HBV transgenic mice model (Chisari 1995), there are still some different levels of flaws.
Dracocephalum heterophyllum is a traditional Tibetan medicine growing on Qinghai-Tibet Platean with special living environment of high elevation and strong sunlight irradiation. The plant is distributed widely in Sitsang, Qinghai, Sinkiang and Gansu province of China. In traditional Tibetan Medicine, D. heterophyllum is known as Ao-Ga or Ji-Mei-Qing-Bao, which has been used as an ethnomedicine to treat various ailments such as jaundice, hepatopathy, cough, lymphangitis, mouth ulcers and tooth diseases. There had been reports said that the herb had antiviral activity (Zhang et al. 2009), antianoxic effect (Peng 1984), antiasthmatic, anticoughing and disinfectant action (Mahmood et al. 2005), and the essential oil of it also had antimicrobial and antioxidant activities (Zhang et al. 2008).
The aim of this paper was to evaluate the anti-hepatitis activities of D. heterophyllum in the mouse fulminant hepatitis model induced by concanavalin A (ConA), and measured the antioxidant activities of this herbs in a series of in vitro assay such as free radical scavenging experiments (DPPH and ABTS assay), anti-lipidperoxidation experiments (FTC assay), ferric reducing antioxidant power assay (FRAP), metal chelating assay and determination of total phenolic contents (TPC). Finally, the bioactive substances were also separated and purified using chromatographic techniques.
Female Bal B/C mice were bought from Beijing Vitalriver Experimental Animals Ltd. (Beijing, China), The animals were performed according to guidelines laid down by the Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (Approval IDs: SCXY2012-0119) that follows internationally acceptable standards on animal care and use in laboratory experimentation. Concanavalin A (ConA) was obtained from Sigma-Aldrich Co. (Shanghai, China), ALT kit, AST kit, TUNEL kit, DAPI kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), linoleic acid, α-Tocopherol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), potassium persulfate (K2S2O8), ammonium thiocyanate were (NH4SCN), ferrous chloride tetrahydrate (FeCl2·4H2O), Ferric chloride tetrahydrate (FeCl3·6H2O), sodium tungstate dihydrate (Na2WO4·2H2O), sodium molybdate dehydrate (Na2MoO4·2H2O), sodium carbonate anhydrous (Na2CO3) were all purchased from Aladdin Industrial Corporation (Shanghai, China). Gallic acid was obtained from National Institute for Food and Drug Control (Beijing, China). All other chemical reagent and buffer used were analytical grade and obtained from Beijing Chemical Co. (Beijing, China).
The whole grass of D. heterophyllum was harvested in August 2014, from North Mountain in Huzhu, Qinghai province, China. The sample was identified by MEI Li-juan (Northwest Institute of Plateau Biology, Qinghai, China). The fresh samples were air-dried in hade, then ground into a homogeneous powder in a mill.
8.86 kg of air-dried and powered D. heterophyllum was extracted by 95 % ethanol at 70 °C through heating reflux. The samples were filtered with filter paper while the residue was further extracted under the same conditions 3 times. The filtrates were collected, and then ethanol was removed by a rotary evaporator (EYELA, Japan) at 50 °C to get the crude extract of D. heterophyllum.
The crude ethanol extract (1026 g) of D. heterophyllum was suspended into 500 mL water. The suspension was successively extracted 3 times by the same volume of petroleum ether, ethyl acetate and n-butanol at room temperature to get four fractions. Then the four fractions were dried by a rotary evaporator (EYELA, Japan), respectively. The four extracts were stored at 4 °C until used.
Anti-hepatitis activity assay
The survival experiment of mice with lethal doses of ConA
Concanavalin A (ConA) is a plants lectin, and is known for its ability to stimulate mouse T cell subsets giving rise to four functionally distinct T-cell populations, including precursors to suppressor T-cell; one subset of human suppressor T-cells as well is sensitive to ConA (Dwyer and Johnson 1981). Female BALB/c mice would develop severe liver injury as assessed by transaminase release within 8 h when an intravenous dose concanavalin A (Con A) was given. Histopathologically, only the liver was affected. Con A-induced liver injury depends on the activation of T lymphocytes by macrophages in the presence of ConA (Tiegs et al. 1992). The model might allow the study of the pathophysiology of autoimmune hepatitis and viral hepatitis.
Female Balb/C mice 9–10 weeks old weighing about 25–29 g were housed for 1 day to acclimatize. The mice were randomly divided into nine groups each comprised of eight mice. The first group, ConA group, was only injected with 1 mg/mL ConA in caudal vein at the lethal dose of 20 mg/Kg. The next four groups, drug group, were only injected with 10 mg/mL four extracts of D. heterophyllum in abdominal cavity at the dose of 50 mg/Kg, respectively. The last four groups, ConA+ drug group, were injected with 10 mg/mL four extracts of D. heterophyllum in abdominal cavity at the dose of 50 mg/Kg, respectively, and 2 h later, were injected with 1 mg/mL ConA in caudal vein at the dose of 20 mg/Kg to induced hepatitis. The mice were fed and observed for 24 h to determine their mortality.
The level of transaminase in mice serum
The treatment on the hepatitis model induced by conA
The number of mice
Method of administration
ConA+ drug group
Caudal vein + abdominal cavity
Morphologic, histological and hepatocytes apoptosis analysis
Pretreating the mice base on the (Table 1). Test mice breaking the neck to death after induced by ConA 24 h later, removing the liver, observing the hepatic pathological changes of liver using morphologic method. Then the livers were fixed in 10 % formalin, embedded in paraffin and cut into slices. HE staining were performed and the result of the staining was analyzed with microscopic examination. The method of TdT mediated dUTP nick end labeling (TUNEL) and 4′,6-diamidino-2-phenylindole (DAPI) were used to detect apoptosis of mouse liver.
DPPH free radical scavenging activity
where AControl is the absorbance of the control reaction, while ASample is the absorbance at 517 nm with D. heterophyllum extracts.
ABTS free radical scavenging activity
where AControl is the absorbance of the control reaction, while ASample is the absorbance at 734 nm with D. heterophyllum extracts.
Anti-lipidperoxidation activity by FTC
where AControl is the absorbance of the control reaction and ASample is the absorbance at 500 nm with D. heterophyllum extracts or standards compounds.
Ferric reducing antioxidant power assay (FRAP)
The ability to reduce ferric ions was measured according to a modified method developed by Benzie and Strain (1996). To prepare the FRAP reagent, 0.1 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl3·6H2O were mixed together in a ratio of 10:1:1 (v/v/v). A 100 μL aliquot of sample solutions (100–500 μg/mL) were added to 3 mL freshly prepared FRAP reagent. The absorbance of the reaction mixture was measured at 593 nm after 10 min incubation at 37 °C. Experiments were performed in triplicate. Aliquots of α-tocopherol, BHT and BHA was served as standards for the assay. The FeSO4·7H2O solutions (0.2–2 mM/L) was used to performed the calibration curves. The ferric reducing antioxidants power were calculated from the linear calibration curve and expressed by FRAP value: 1FRAP value = 1 mmol/L FeSO4.
Ferrous metal ions chelating activity
where AControl is the absorbance of the control reaction, while ASample is the absorbance at 562 nm with D. heterophyllum extracts. The control contains FeCl2 and ferrozine, complex formation molecules.
Determination of total phenolic contents (TPC)
The total phenolic contents of four D. heterophyllum extracts were determined using the Folin-Ciocalteu method (Ragazzi and Veronese 1973) with a little modification. To prepare the Folin-Ciocalteu reagent, 25 g sodium tungstate, 6.25 g sodium molybdate, 175 mL distilled water, 8.5 % phosphoric acid solution, and 25 mL concentrated hydrochloric acid were mixed together. The above mixture was refluxed slowly in a water bath for 10 h. After cooling, 37.5 g lithium sulfate, 12.5 mL distilled water, and 50 mL hydrogen peroxide were added in, then continued to heat in boiling water for 15 min without cap. Finally dilute with water to 250 mL, and stored at 4 °C until used. 100 μL sample solution (1 mg/mL) was mixed with 500 μL Folin-Ciocalteu reagent and diluted with 1000 μL distilled water, then 1.5 mL of Na2CO3 solution (20 %, w/v) was added. Absorbance of the mixture was measured at 765 nm after 2 h incubation in dark at home temperature. The determinations were performed in triplicate, and the calculations were based on a calibration curve obtained with gallic acid (100–800 μg/mL).
Separation and purification of the bioactive compounds
The petroleum ether extract (500 g) was subjected to a silica gel column (petroleum ether-acetone, 95:5–5:95) to afford fractions (I–VI). Fraction III (150 g), the mixture of two compounds, was purified on a preparative C18 HPLC column with a isocratic of MeOH–H2O (87:13) to yield compound 1 (3 mg) and compound 2 (33 mg). The two compounds were difficult to separate. Fraction IV was purified on a preparative C18 HPLC column with a gradient of C2H3N–H2O (65:35–95:5) to yield compound 3 (130 mg) and compound 4 (75 mg). Fraction VI was purified on a preparative C18 HPLC column with a gradient of C2H3N–H2O (20:80–30:70) to yield compound 5 (200 mg).
The ethyl acetate extract (19 g) was subjected to medium pressure liquid chromatography with a gradient of MeOH–H2O (3:7–8:2) to afford fractions (I–VII). Fraction III (3 g) was purified on a preparative C18 HPLC column with a gradient of C2H3N−H2O (10:90–30:70) to yield compound 6 (1.5 g). Fraction IV (2.3 g) was purified on a preparative C18 HPLC column with a isocratic of C2H3N−H2O (25:75) to yield compound 7 (853 mg). Fraction V (1.1 g) was purified on a preparative C18 HPLC column with a isocratic of C2H3N−H2O (28:72) to yield compound 8 (58 mg) Fraction IV (1.1 g) was purified on a preparative C18 HPLC column with a isocratic of C2H3N–H2O (27:73) to yield compound 9 (87.9 mg).
Results and discussion
Extraction and fractionation of Dracocephalum heterophyllum
From 8.86 kg of D. heterophyllum, 1026 g of 95 % ethanol extract was obtained. The yield was 11.6 %. The crude extract (1026 g) was then suspended in water and extracted with petroleum ether, ethyl acetate and n-butanol sequentially to get 500 g petroleum ether fraction (48.7.6 %), 19 g ethyl acetate fraction (2 %), 41.8 g n-butanol fraction (4.1 %) and 115.3 g water fraction (11.2 %).
The survival experiment of mice with lethal doses of ConA
The level of transaminase in mice serum
Morphologic, histological and hepatocytes apoptosis analysis
Liver damage caused by hepatitis is associated with excessive activation of the immune responses. It has been reported that TNF-α participate in various forms of liver damage, such as viral, toxic and autoimmune hepatitis, and play an important role in ConA-induced hepatitis. It has also been reported that Kupffer cells secrete a large amount of TNF-α to aggravate the liver damage. Kupffer cells play an important role in T cell activation-induced liver injury. However, the mechanism may not be that simple, and more research is needed.
DPPH free radical scavenging activity
ABTS·+ free radical scavenging activity
Ferric reducing ability of plasma (FRAP)
The FeSO4·7H2O solutions (0.2–2 mM/L) was used to performed the calibration curves, regression equations of it was Y = 0.68006X + 0.00916, R2 = 0.999. The ferric reducing antioxidant power of four D. heterophyllum extracts and standards antioxidants were expressed as FRAP value: 1 FRAP value = 1 mmol/L FeSO4.
Ferrous metal ions chelating activity
Total phenolic contents of fractions of Dracocephalum heterophyllum
By way of the literature review (Li et al. 2012), total phenolic was of significant positive correlations with antioxidant levels. In this paper, the total phenolic contents of petroleum ether, EtOAc, n-BuOH and water fractions were measured by the Folin-Ciocalteu reagent. The gallic acid (100–800 μg/mL) was used to performed the calibration curves, the regression equations of it was Y = 0.001X + 0.1693, R2 = 0.9957, and the total phenolic contents was expressed as Gallic acid mg/g of dry material. The results showed that EtOAc fraction had the highest phenolic contents of 433.7 mg gallic acid/g, n-BuOH and petroleum ether fraction had the phenolic contents of 110.7 and 70.7 mg/g. The water fraction had the lowest phenolic contents of 31.7 mg Gallic acid/g of dry material. This results indirectly reflect the antioxidant activities of Dracocephalum heterophyllum extracts.
The correlation of total phenolic contents (TPC) with DPPH assay (R2 = 0.9637, P < 0.05), ABTS assay (R2 = 0.9638, P < 0.05), FTC assay (R2 = 0.8203, P < 0.05) and FRAP assay (R2 = 0.9991, P < 0.05), respectively. The results demonstrated that the antioxidant activities of D. heterophyllum extracts had high correlation with total phenolic contents (TPC).
The chemical constituent of Dracocephalum heterophyllum
In this paper, the mouse fulminant hepatitis model induced by ConA was first used to research the anti-hepatitis activity of petroleum ether, ethyl acetate, n-butyl alcohol and water extracts of D. heterophyllum. The antioxidant activity was also studied by some experiment in vitro, and the bioactive substances were isolated using chromatographic techniques and identified by NMR spectroscopic methods. Our results indicated that the ethyl acetate extract of D. heterophyllum had the highest anti-hepatitis and antioxidants activities, followed by the petroleum ether extract. The bioactive substances may be triterpenes, flavonoids and phenolic acids, the ethyl acetate extracts of D. heterophyllum may be possible candidates in developing anti-hepatitis medicine.
Q-QS principal conducted the experiment and wrote the manuscripts. Associate professor Q-LW helped us to modify the manuscripts. All authors participated in drafting the manuscript. All authors read and approved the final manuscript.
The authors gratefully appreciated the support by senior engineer Li-Juan Mei, from Northwest Institute of Plateau Biology, CAS, who helped us identifying the authenticity of D. heterophyllum. The authors also like to thank the grant support from Advanced technology research and development program of Qinghai Province (2014-GX-220).
The authors declare that they have no competing interests.
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