Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian)

Andrographis paniculata (Burm. f.) Nees (Acanthaceae) is a medicinal plant used in many countries. Its major constituents are diterpenoids, flavonoids and polyphenols. Among the single compounds extracted from A. paniculata, andrographolide is the major one in terms of bioactive properties and abundance. Among the andrographolide analogues, 14-deoxy-11,12-didehydroandrographolide is immunostimulatory, anti-infective and anti-atherosclerotic; neoandrographolide is anti-inflammatory, anti-infective and anti-hepatotoxic; 14-deoxyandrographolide is immunomodulatory and anti-atherosclerotic. Among the less abundant compounds from A. paniculata, andrograpanin is both anti-inflammatory and anti-infective; 14-deoxy-14,15-dehydroandrographolide is anti-inflammatory; isoandrographolide, 3,19-isopropylideneandrographolide and 14-acetylandrographolide are tumor suppressive; arabinogalactan proteins are anti-hepatotoxic. The four flavonoids from A. paniculata, namely 7-O-methylwogonin, apigenin, onysilin and 3,4-dicaffeoylquinic acid are anti-atherosclerotic.

Andrographolide exhibits multiple pharmacological properties and is a potential chemotherapeutic agent [19]. Andrographolide contains an α-alkylidene γ-butyrolactone moiety and three hydroxyls at C-3, C-19 and C-14 responsible for the cytotoxic activities of andrographolide against many cancer cell lines [19]. Andrographolide is abundant in leaves and can be easily isolated from the crude plant extracts as crystalline solid [5,10,17,20,21].

Pharmacological properties
A. paniculata exhibits a vast range of pharmacological properties (Tables 2 and 3).

Anti-inflammation effects
Systemic inflammation was suggested to be associated with increased risk of chronic diseases such as cardiovascular disease, cancer and insulin resistance [22]. Inflammation involves macrophage and T lymphocyte activation as well as the release of pro-inflammatory mediators, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, interferon (IFN)-γ, nitric oxide (NO) and cell adhesion molecules which in turn amplify the inflammation [23]. Effective modulation of the aberrant production of these molecules may reduce inflammation [24,25].
Neoandrographolide, isolated from EtOAc portion in methanol extract, suppresses NO production both in vitro and ex vivo in bacillus Calmette-Guéin (BCG)induced peritoneal macrophages [34] in mice. Neoandrographolide inhibits in vitro TNFα and PGE 2 production in RAW cells, suppresses ear oedema induced by dimethyl benzene in mice [35,36]. Andrograpanin, a hydrolysate from neoandrographolide, reduces NO, TNFα and IL-6 production in LPS-activated macrophage cells derived from bone marrow in mice, possibly due to down-regulation of p38 mitogen-activated protein kinase (MAPKs) signalling pathways [37].

Anti-cancer effects
Kumar et al. fractionated the methanol extract of A. paniculata into dichloromethane, petroleum ether and aqueous extracts and found that only the dichloromethane fraction significantly inhibited the proliferation of HT-29 colon cancer cells [41]. They further fractionated the dichloromethane extract and yielded three diterpene compounds, namely andrographolide, 14-deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide. Andrographolide showed the greatest anti-cancer activity on a range of cancer cells [41]. The A. paniculata ethanol extract showed cytotoxic activities against human cancer cell lines, such as Jurkat (lymphocytic), PC-3 (prostate), HepG2 (hepatoma) and Colon 205 (colonic) cells [42]. An in vivo study demonstrated that A. paniculata 70% ethanol extract and andrographlide increased the life spans of mice injected with thymoma cells [43]. Isolated from 85% ethanol extract of A. paniculata, andrographolide and isoandrographolide exhibited higher antiproliferative activities in human leukaemia HL-60 cells than other 16 ent-labdane diterpenoids with IC 50 's of 9.33 and 6.30 μM respectively [44].
The anti-cancer mechanisms of andrographolide have been investigated [19]. Andrographolide and its analogues exert direct anti-cancer activities on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein and decreased expression of cyclin-dependent kinase [45][46][47][48][49]. Other compounds may block the cell cycle progression at G2/M phase [42]. Andrographolide inhibits human hepatoma cell growth through activating c-Jun N-terminal kinase [50] or inducing cell differentiation [51]. Andrographolide induces apoptosis in human cancer cells via the activation of caspase 8, pro-apoptotic Bcl-2 family members Bax conformational change, release of cytochrome C from  ethanol extract serum anti-Salmonella typhinurium IgG levels IFN-γ in Con A-stimulated splenocytes of mice (orally, 25 or 50 mg/kg BW, 14d) [76] antibody and the delayed-type hypersensitivity response (orally 25 mg/kg, 7d) [74] G0/G1 phase mitochondrial CYP and expression of Bax in human leukemic HL-60 cells [49] expression of EBV lytic proteins during the viral lytic cycle in P3HR1 cells [82] fasting serum glucose in diabetic rats (orally 0.1, 0.2, and 0.4 g/BW, 14d) liver and kidney TBARS levels liver GSH concentrations (orally 400 mg/kg BW, 14d) [113] 95% ethanol extract RANTES secretion by human bronchial epithelial cells infected with influenza A virus H1N1 [86] 80% ethanol extract hepatic GPX, GR, CAT, SOD; lipid peroxidation (orally 50, 100 mg/kg BW, 14d) [121] 70% ethanol extract CTL production through enhanced secretion of IL-2 and IFNγ by EL-4 T cells [43] serum NO, VEGF and TIMP-1, angiogenesis in melanoma cell implanted mice (i.p. 10 mg/d, 5d) [56] 95% ethanol or EtOAc extract pi class of glutathione S-transferase expression in rat primary hepatocytes [99] EtOAc extract NF-κB trans-activation assayed by NF-κB-dependent luciferase activity ex-vivo NO and PGE 2 production by LPS/IFN-γ-stimulated peritoneal macrophages LPS-induced acute inflammation in mice (orally 0.78~3.12 mg/kg BW, 7d) [5,39] aqueous extract protect nicotine-induced toxicity in brain (i.p. 250 mg/kg BW, 7d) nicotine induced DNA fragmentation in lymphocytes, lipid peroxidation, protein oxidation [93,92] systolic blood pressure of SHR and WKY rats (i.p. 0.7, 1.4, 2.8 g/kg BW) [101] blood glucose in STZ-induced hyperglycaemic rats (50 mg/kg BW, 10d) [115] hepatic CAT, SOD and GST activities in lymphoma bearing mice (orally 10~30 mg/d) [123] d: day; BW: body weight mitochondria and activation of caspase cascade [52] and/ or via the activation of tumour suppressor p53 by ROSdependent c-Jun NH 2 -terminal kinase (JNK) activation, thereby increasing p53 phosphorylation and protein stabilization [53,54]. Andrographolide may suppress an oncogene v-Src-induced transformation and down-regulate v-Src protein expression via the attenuation of ERK1/ 2 signalling pathway [55]. In addition, enhancement of immunity and inhibition of angiogenesis and tumour cell migration may also contribute to the anti-cancer effects. Inhibiting human cancer cell growth, A. paniculata extract enhances proliferation and IL-2 induction in human peripheral blood lymphocytes [41]. Sheeja et al. showed that the A. paniculata ethanol extract and andrographolide stimulated the cytotoxic T lymphocytes (CTL) activity through enhanced release of IL-2 and IFNγ in serum thereby inhibiting tumour growth [43]. The A. paniculata ethanol extract and andrographolide successfully inhibited the tumour specific capillary sprouting without damaging the pre-existing vasculature in mice injected with melanoma cells. A. paniculata extract inhibits tumour specific angiogenesis by down-regulating various proangiogenic molecules such as vascular endothelial growth factor (VEGF), NO and proinflammatory cytokines and up-regulating anti-angiogenic molecules such as IL-2 and tissue inhibitors of metalloproteinase-1 (TIMP-1) which prevent tumour metastasis [56]. As tumour cells can express high levels of sialyl Lewis surface antigens that interact with adhesion molecules E-and P-selectins on activated endothelial cells, cancer cell adhesion to endothelial cells followed by tumour extravasation results in metastasis. Andrographolide inhibits the adhesion of cancer cells to the activated endothelium by blocking E-selectin expression [57]. Andrographolide may also inhibit angiogenesis for tumour metastasis via down-regulating matrix metal-loproteinases-7 (MMP-7) expression, possibly by inactivating activator protein-1 (AP-1) through suppressing PI3K/Akt signalling pathway [58,59].
A novel semi-synthetic analogue of andrographolide, DRF3188, shows anti-cancer activities at a lower dosage than andrographolide through a similar mechanism [46]. Synthesis and structure-activity relationships of andrographolide analogues as novel cytotoxic agents reveals that intact α-alkylidene γ-butyrolactone moiety of andrographolide, the D12(13) double bond, the C-14 hydroxyl or its ester moiety and the D8(17) double bond or epoxy moiety are responsible for the cytotoxic activities exhibited by andrographolide and its analogues [60]. Anti-cancer agents usually possess selective growth inhibition or cytotoxic properties [61]. The semi-synthesized andrographolide derivatives were screened against a panel of 60 human cancer cell lines. The results showed that 3,19-isopropylideneandrographolide was selective towards leukaemia and colon cancer cells whereas 14acetylandrographolide was selective towards leukaemia, ovarian and renal cancer cells [47]. The benzylidene derivatives of andrographolide showed more potent anticancer activities than andrographolide [62]. The addition of andrographolide to 5-Fluorouracil induces synergistic apoptosis [54]. Moreover, andrographolide enhances the sensitivity of cancer cells to a chemotherapeutic drug, namely doxorubicin, mainly via suppressing JAK-STAT3 [63]. The results of these studies suggest a potential therapeutic strategy of combining andrographolide with chemotherapeutic agents to treat cancer.

Immunomodulatory effects
Immune responses such as proliferation of lymphocytes, antibody production and cytokines secretion are regulated under normal conditions. Every immunocompetent cell is controlled by other cells with antagonistic action [64]. The balance between type 1 T helper cell-mediated and type 2 Th cell-mediated immune responses is critical for immunoregulation.
A. paniculata dichloromethane extract significantly augments the proliferation of human peripheral blood lymphocytes (hPBL) at low concentrations [41]. The three diterpene compounds including andrographolide enhance proliferation and IL-2 secretion in hPBL [41]. Andrographolide enhances secretion of IL-2 and IFNγ by T cells and stimulates the production of cytotoxic T lymphocytes [43,65].
On the other hand, when murine T cell is stimulated with mitogen, IL-2 was decreased by andrographolide [66] possibly via reducing nuclear factor of activated T cells (NFAT) activities and increasing JNK phosphorylation [67]. Similarly, andrographolide interferes with T cell activation and reduces the severity of experimental autoimmune encephalomyelitis (EAE) in mice. Clinical signs of disease such as abnormal gait and limb paralysis, pro-liferation and IL-2 secretion of lymph node cells, as well as cell-dependent antibody production in EAE mice were reduced by andrographolide treatment [68]. Andrographolide is beneficial for inflammation-related immune dysregulatory diseases, such as allergic asthma, rheumatoid (RA) and neurodegenerative diseases via inhibition of the NF-κB signalling pathway [69]. Andrographolide reduces inflammation-mediated dopaminergic neurodegeneration in mesencephalic neuron-glial cultures by inhibiting microglial activation and production of proinflammatory factors such as TNFα, NO and PGE 2 [70]. Andrographolide inhibits OVA-induced increases in total cells, eosinophils and IL-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF), and reduces serum level of OVA-specific IgE [71]. Andrographolide attenuated OVA-induced lung tissue eosinophils and airway mucus production, mRNA expression of E-selectin, chitinases, mucin Muc5ac and iNOS in lung tissues and airway hyperresponsiveness [71]. Andrographolide inhibits OVA-induced increases TNF-α and GM-CSF in BALF of OVA-sensitized and nasally-challenged mice [72]. A recent clinical study showed that A. paniculata extract (30% andrographolide) reduced the symptoms and certain immunological parameters such as serum immunoglobulins and complement components in patients with rheumatoid arthritis during a 14-week treatment [73].
Oral administration of the A. paniculata ethanol extract or andrographolide to mice stimulated antibody production and the delayed-type hypersensitivity response to sheep red blood cells [74]. Andrographolide increases spontaneous IFNγ and mitogen-stimulated TNF-α secretion by cultivated human peripheral blood cells [75]. Oral pre-treatment of the A. paniculata ethanol extract or andrographolide in mice immunized with an inactivated Salmonella typhimurium vaccine enhances Salmonella-specific IgG antibody and IFN-γ production [76]. Recent study demonstrated that the cyclophosphamide-potentiated DTH reaction was reversed by a pure powder mixture of andrographolide plus 14-deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide together. The mixture stimulated phagocytosis, and elevated antibody titer and plaque-forming cells in the spleen cells in mice [77].

Anti-Oxidation
MDA formation [91] GSH, SOD activity [92,93] [79]. A phase I dose-escalating clinical trial of andrographolide in HIV positive patients reported a significant rise in the mean CD4 + lymphocyte level of HIV patients. Andrographolide inhibits HIV-induced cell cycle dysregulation, leading to a rise in CD4 + lymphocyte levels in HIV-1 infected individuals [80]. Andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide isolated from A. paniculata demonstrated viricidal activity against herpes simplex virus 1 (HSV-1) without significant cytotoxicity [81]. The A. paniculata ethanol extract and andrographolide inhibit the expression of Epstein-Barr virus (EBV) lytic proteins during the viral lytic cycle in P3HR1 cells, an oral lymphoma cell line latently infected by EBV. Andrographolide inhibits the production of mature viral particles and is not toxic to P3HR1 cells [82].
A recent in vitro study investigated the anti-influenza activity of A. paniculata in canine kidney cell line as well as mice infected with H1N1, H9N2 or H5N1 [83]. A newly synthesized andrographolide derivative 14-α-lipoyl andrographolide was more effective against avian influenza A (H9N2 and H5N1) and human influenza A H1N in vitro than andrographolide [83]. Another andrographolide analogue 14-glycinyl andrographolide hydrochloride inhibits virulence factor production and bacterial growth [84]. Moreover, a double blind, placebo-controlled, parallel-group clinical study on a combined formula of A. paniculata extract and Acanthopanax senticocus, also known as Kan Jang, demonstrated the formula's positive effects in treating acute upper respiratory tract infections and relieving the inflammatory symptoms of sinusitis [85].
The migratory response of leukocytes to chemokines forms the first line of defence to the invading microbial. A. paniculata extract inhibits secretion of RANTES, a potent chemoattractant exacerbating bronchial inflammation as a result of H1N1-infected human bronchial epithelial cells [86]. Andrograpanin enhanced chemokine stromal cell-derived factor-1α (SDF-1α) induced chemotaxis in Jurkat and THP-1 cells via CXC chemokine receptor-4 specific induced cell chemotaxis [87]. Andrograpanin enhancing chemokine-induced leukocyte chemotaxis may contribute to the anti-infectious function of A. paniculata. Post-translational cleavage by proprotein convertase is one of the several events that determine the viral infectivity and virulence [88]. The inhibitory action of andrographolide was enhanced significantly by the formation of dehydroandrographolide succinic acid monoester (DASM) via succinoylation [88]. DASM inhibits HIV by interfering with HIV-induced cell fusion and with HIV's binding to the cell [89].

Anti-hepatotoxic effects
Liver metabolizes xenobiotics, such as drugs, toxins and chemical carcinogens; chronic liver injury leads to cirrhosis. Anti-hepatotoxic enzymes include cytochrome P450 (CYP) super-family, or normalizing the levels of marker enzymes for the liver function test, such as glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), acid phosphatase (ACP) and alkaline phosphatase (ALP) [90].
An early study showed that pre-treatment of mice with andrographolide, andrographiside and neoandrographolide alleviated hepatotoxicity induced by intoxication of carbon tetrachloride (CCl 4 ) or tert-butylhydroperoxide (tBHP) in mice [91]. The glucoside groups in andrographolid and neoandrographolide were suggested to act as strong antioxidants. The A. paniculata aqueous extract and andrographolide decreased oxidative stress in isolated rat lymphocytes exposed to nicotine [92]. The A. paniculata aqueous extract and andrographolide ameliorated the dysfunction in the brain associated with nicotine toxicity [93]. Arabinogalactan, another aqueous component of the A. paniculata, Tris-buffer extract and andrographolide minimized the toxicity in pre-treated mice [90]. Oral treatment of rats with the A. paniculata methanol extract followed by CCl 4 administration restored plasma lipid peroxidation, alanine transaminase (ALT) and aspartate transaminase (AST) [94].
Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentrationdependent manner, and synergistically induced CYP1A1 expression with the typical CYP1A inducers [95,96]. In addition, the A. paniculata 60% ethanol extract or andrographolide may cause herb-drug interactions through CYP3A and CYP2C9 inhibition in vitro or CYP2C11 inhibition in vivo [97,98]. Induction of drugmetabolizing enzymes is considered to be an adaptive response to a cytotoxic environment. The A. paniculata ethanol extract, EtOAc extract and andrographolide induce the expression of the pi class of glutathione Stransferase, a phase II biotransformation enzymes involved in detoxification of various classes of environmental carcinogens, in rat primary hepatocytes [99]. A recent study showed that this induction by andrographolide was suppressed by the increase of cAMP or cAMP analogues [100].

Anti-atherosclerotic effects
Zhang et al. reported that the A. paniculata aqueous extract lowers systolic blood pressure (SBP) of both spontaneously hypertensive rats (SHR) and the control Wistar-Kyoto rats [101]. The A. paniculata water, nbutanol and aqueous extracts produce a significant fall in mean arterial blood pressure (MAP) without significant decrease in heart rate in anaesthetized Sprague-Dawley rats [102]. The 14-deoxyandrographolide isolated from the A. paniculata methanol extract showed vasorelaxant effects in isolated rat thoracic aorta [103]. Another diterpenoid isolated from A. paniculata methanol extract, 14deoxy-11,12-didehydroandrographolide, significantly reduces MAP and heart rate and beating rate of isolated right atria in anaesthetised rats [104]. These two diterpenoids may exert their vasorelaxant activities through the activation of the NOS and guanylyl cyclase for NO release from endothelial cells [105]. Moreover, 14deoxyandrographolide reduces the contractile response by calcium channel-dependent rat uterine smooth muscle contraction [106] The vascular smooth muscle is the major site of the hypotensive effects of the A. paniculata hot water extract and 14-deoxy-11,12-didehydroandrographolide [107], suggesting relaxant effects of A. paniculata in muscle.

Anti-hyperglycaemic effects
Hyperglycaemia is involved in the aetiology of development of diabetic complications. Hypoglycaemic herbs increase insulin secretion, enhance glucose uptake by adipose or muscle tissues and inhibit glucose absorption from intestine and glucose production from liver [111]. Oral administration of the A. paniculata ethanol extract significantly reduced the fasting serum glucose level in streptozotocin (STZ) induced diabetic rats. No significant change in insulin level was observed among the  NF-κB trans-activation activity was assay as following: RAW 264.7 macrophages transiently transfected with NF-κB reporter plasmids were pretreated various compounds and then stimulated with LPS 100 ng/mL/IFN-γ 1000 units/mL for further was estimated by the Dual-Glo Luciferase reporter assay. The collected cell supernatants were assayed for TNFα, IL-6, and MIP-2 productions using commercial ELISA kits. NO was determined by Griess assay [17].
three groups of diabetic rats. The activity of hepatic glucose-6-phosphalase (G-6-Pase) and fasting serum triglyceride levels were significantly reduced by the A. paniculata extract [112]. In addition to its hypoglycaemic action, the A. paniculata may also reduce oxidative stress in diabetic rats [113]. An in vitro study and in vivo oral carbohydrate tolerance tests in STZ-induced diabetic rats suggest that α-glucosidase inhibition may be responsible for the anti-diabetic activity of A. paniculata ethanol extract [114]. The A. paniculata aqueous extract significantly reduces blood glucose in hyperglycaemic rats without significantly changing the rats' weight [115]. However, alloxaninduced diabetic rats treated with A. paniculata water extract had higher body weight than the positive (diabetic) controls; the blood glucose levels were significantly reduced and impaired oestrous cycle in alloxan-induced diabetic rats was restored [116].
Andrographolide significantly attenuated the increase of plasma glucose induced by an intravenous glucose challenge test in normal rats. Andrographolide enhanced the uptake of glucose and the mRNA and protein levels of the glucose transporter subtype 4 (GLUT4) in soleus muscle in STZ-diabetic rats [117]. Andrographolide not only reduced expression of phosphoenolpyruvate carboxykinase (PEPCK) in liver of STZ-diabetic rats, activated α1-adrenoceptors to enhance the secretion of βendorphin, thereby stimulating the opioid μ-receptors to reduce hepatic gluconeogenesis and to enhance the glucose uptake in soleus muscle, leading to a decrease of plasma glucose in STZ-diabetic rats [118]. A recent study showed that andrographolide-lipoic acid conjugate (an andrographolide analogue) had both hypoglycaemic and beta cell protective effects [119].

Anti-oxidative activities
Antioxidant action is manifested by a decrease of malondialdehyde (MDA) formation via lipid peroxidation and an increase of hepatic antioxidative enzymes and antioxidants such as glutathione peroxidase (GPX), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and glutathione S transferase (GST). Anti-oxidative activity of A. paniculata contributes to its antiinflammatory, anti-cancer, anti-hepatotoxic, anti-atherosclerotic and anti-diabetic activities [27,44,91,108,117].
An in vitro scavenging of superoxide radical assay suggests that neoandrographolide from the A. paniculata hexane/EtOA C extract is an effective in vivo scavenger for small radicals [120].
An in vivo study demonstrated that the A. paniculata 80% ethanol extract enhanced murine hepatic antioxidative enzymes and antioxidants such as GPX, GR, CAT and SOD but reduced lipid peroxidation [121]. The A. paniculata methanol extract significantly lowered MDA levels and raised the total antioxidant status in urine samples 24 hours after oral administration [122]. The A. paniculata methanol extract preserved CAT and SOD activities in erythrocytes after CCl 4 administration [94]. Oral administration of the A. paniculata aqueous extract significantly enhanced CAT, SOD and GST activities in the liver of lymphoma bearing mice [123]. Moreover, the A. paniculata aqueous extract exhibited more antioxidant action than its ethanol extract in terms of free radical scavenging, xanthine oxidase inhibition and anti-lipid peroxidation [124].