Andrographolide

Review

1.

Introduction

2.

Sites of modification

3.

Modification for anticancer

Chantana Aromdee

4.

Modification for

Khon Kaen University, Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen, Thailand

neuroprotective

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Khon Kaen University on 09/18/14 For personal use only.

Andrographolide: progression in its modifications and applications -- a patent review (2012 -- 2014)

5.

Anti-dyslipidemic, low-density lipoprotein-oxidation and antioxidant activities

6.

Antivirals

7.

Anti-inflammatory

8.

Interaction of Androg and Andrographis paniculata dried extract with drugs

9.

Androg patent update

10.

Conclusion

11.

Expert opinion

Introduction: Extraction, isolation and modifications of andrographolide (Androg) is extensively investigated and patented. The prominent activities were vastly modified for anticancer and antivirals. Many products related to Androg are commercially available, thus the section ‘Interaction of Androg and Andrographis paniculata dried extract with drugs’ is included. Areas covered: The data in this review are searched and selected from SciFinder and Espacenet for the patents, with the keywords: Andrographolide and Andrographolide analogs, and the results were refined by the years. Expert opinion: Modifications of Androg have been done to nearly all of the possible sites, and now screening tests for any new activities had been settled down. Categorizing the analogs that have been developed is not clear cut since some diseases can develop into others, for example, inflammation and some viral infections can develop into cancer. Currently, investigation of the mode of action and the mechanisms at the molecular level are intensively ongoing. Producing new chemotherapeutic agents from Androg looks promising. The main problem of using Androg in therapeutic applications is its insolubility in aqueous media. Those modified analogs’ esters, ethers or salts, have to be considered for the stability of pharmaceutical preparations, and transformation in biological fluids after administration. Further stages of drug development are required for those promising analogs. Keywords: analogs, andrographolide, anticancer, anti-inflammatory, modification, therapeutic patent Expert Opin. Ther. Patents (2014) 24(10):1129-1138

1.

Introduction

Andrographis paniculata, a traditional herbal medicine, is used in India (Ayurveda and Unani), China and throughout Asian countries. The herb is official in pharmacopoeia of many countries, that is, the United States Pharmacopeia (USP) 36 as a dietary supplement [1] and Thai Herbal Pharmacopoeia [2]. The total diterpene content specified in the USP is 1%, which is equal to that specified in Indian Pharmacopoeia 1966 [3], whereas the Thai’s is 6%. However, the assay methods are different. Dried extract of the herb is a monograph in both USP’s and Indian’s. Pharmaceutical preparations of the herb extract are also official in Chinese Pharmacopoeia, such as Chuan Xin Lian tablets and capsules [4]. The extract is now available worldwide in various dosage forms, they were claimed to treat common cold sinusitis and inflammatory and hepatoprotective activities. The requirement to declare the definite contents of the individual or total g-lactone diterpenes, andrographolide (Androg), 14-deoxy-11,12-didehydroandrographolide (14-DDA),

10.1517/13543776.2014.956084 © 2014 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674 All rights reserved: reproduction in whole or in part not permitted

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C. Aromdee

Article highlights. . . . . .

Potential of Andrographolide (Androg) analogs. Trends in modifications of Androg. Androg and its analogs’ activities at the molecular level. Interaction of Androg and Andrographis paniculata extract with drugs. How far are we with production of pharmaceutical preparations from herb/pure compounds.


This box summarizes key points contained in the article.

neoandrographolide, and 14-deoxyandrographolide (14-DA) (Figure 1), depends on the regulations of the countries they are produced. Yanhuning, 14-DDA succinate salts for parenteral use are also included in the Chinese Pharmacopoeia, whereas the method of preparation is patented [5-7]. The second-most main component in A. paniculata is 14-DDA, and is easily transformed from Androg naturally or by reduction with aluminium oxide in pyridine [8]. At present, both Androg and 14-DDA are the main components subjected to modification and expected to increase their original biological activities. The simplest modifications are substitutions of the available OHs to reduce the polarity by esterification or etherification to fit with the target or the permeability through cells. Conjugation with desirable active pharmacophores to enhance their original activities was also extensively investigated [9]. In this review, the activities against various cancer cells, viruses and inflammatory are described as well as their mechanisms. Not many analogs have been investigated through clinical trials; however, patenting analogs are still going on. 2.

Sites of modification

Previously, the most common sites of modification are at the three OHs at C-3, C-14 and C-19, which are easy to etherify, esterify or oxidize to carbonyl. If substituents of these three sites are the same, only a one-step reaction is required. If the 14-OH substitution is different from 3- and 19-OHs’, or sites other than these three OHs are modified, then protection of these OHs is required, since OH is reactive. Very often, intermediates were assessed for the activities as well [10,11]. Androg and 14-DDA are not soluble in water, which is one of the main problems in utilizing these compounds; the solubility of Androg is 1.32 10-4 mol.dm3 at 25 C [12]. Thus, one of the objectives of modification is to make them more water soluble. Dicarboxylic acids, for example, succinic acid substitution at the 3 or 19-OHs or 3,14,19-OHs are commonly made, thus salts can be formed either with sodium or potassium [5]. Additionally, esterification of the carboxylic acid with amino acids, glycyl, L-alanyl and L-leucyl, was also accomplished and used for antiviral, antibacterial infections as well as analgesics and anti-inflammatories [13]. Formation of the OHs to be sulfate or sulfonate or phosphate to enhance the solubility is another attempt [14]. 1130

The double bond at C-8 and C-17, or double bonds at C-12 or C-13 or C-11 and C-13 are also the reactive sites for modification. Epoxidation of the 8,17-double bond along with the modification on other sites is included for anticancer since the outcome of some epoxidated analogs increase the activity [15,16]. Modification at this site was also found for antidyslipidemic [17]. More complicated components such as aromatic, heterocyclic or bulky groups are common [11,13,18], as well as the cycloaddition at C-12 of Androg [19,20] are prone for substitutions of either the same or different constituents at 3,19. Rational or systematic design for modification is not very strong. Factorial design is used for substitution of the 3-OHs. At present, some of modified analogs published before year 2012 were tested for additional activities or mechanisms, some patents were re-patented when the original patens expired. In this review, only active analogs were described. As had been described in the previous review [9], Androg is polar on both ends, the aims of the modification either increase the cell permeability or its ability to fit the target sites. Once an analog/Androg has permeated into cell, binding to various cell constituents especially proteins composed of amino acids and also other polar constituents; interaction with these constituents is unavoidable. Thus, one should be aware that undesirable effects such as interference with the action and metabolism of the conventional therapeutic drugs can occur. 3.

Modification for anticancer

Cancer is an unresolved and worldwide threatening disease and different kind of cancers require different therapeutic treatments. Thus, investigation of Androgs as cancer therapeutics is of great interest. Androg and 14-DDA are cytotoxic to adherent or nonadherent cells differently. Androg, not 14-DDA, was reported to exert cytotoxicity on lung carcinoma (A549), human lung epithelial cells (BEAS-2B), and rat leukemia (KBL)-2H3 [21]. Whereas, 14-DDA is more cytotoxic to human promocytic leukemia (THP-1) cells [22]. Cells for biological activity tests Different cell types contain different gene expression patterns that define cell function. The pattern of expressions can be altered due to the environment, infection and stress from inflammation or epigenetic influence. To determine the anticancer activity of a compound, various cancer cells are used for screening. Once anticancer activity is positive, investigation for the cause of cell death or apoptosis was determined. However, sensitivity is different depending on cell origin. At present, cells used to test for cancer are widely available due to the progress in cell biotechnology. For anticancer activity, cancer cells either nonadherent (i.e., Jurkat, peripheral blood mononuclear cells, THP-1) or adherent cells (i.e., HCT-116, MCF-7), which were isolated from the affected organs, are employed. Cells isolated from laboratories were 3.1

Expert Opin. Ther. Patents (2014) 24(10)

Andrographolide

HO

14

O

O

O O

O (E) 11

3

8

HO 19

HO

HO HO

Andrographolide (Androg)

19

11

3

3

H

O

11

11

4

O O

HO HO

H

14-deoxy-11,12-didehydro andrographolide (14-DDA)

19

3 H

14-Deoxyandro grapholide (14-DA)

GluO

19

H

Neoandrographolide


Figure 1. Structures of Androg and its natural derivatives.

A.

B. O HO

Br or CI or I

O 14

O 14 O

N 11

HO O 8 O HO

19 HO

H

3

N H

8 4

O O

19

Cyclization of Androg at C-12 and C-13 Hazra et al., 2012 [19] had synthesized two series of oxindoles (di-spiropyrrolidino- and di-spirolizidino-oxindole), these two series of heterocyclic compounds are formed at the C-12 and C-13 of Androg. Dey et al. [28] tested these analogs for the apoptosis activity and mechanisms on six adherent human cancer cell lines as well as normal human cell lines (Chang liver cell). The 5¢-bromo-substituted pyrrolidine2-spiro-3¢-oxindole- (Figure 2A), is the most potent analog followed by the chloro and iodo derivatives, respectively. Arresting state was at the G1-phase. The molecular mode of action was determined in colorectal carcinoma (HCT116) cells. Upregulation of Bax, Bad, p53, caspase-3, caspase-9 and cleaved PARP; downregulation of Bcl-2, cytosolic NF-kB p65, PI3K and p-Akt; and translocation of P53/ P21, NF-kB p65 were detected. The GI50 of Androg is > 40 µM on HCT116. 3.3.1

H

Br

Figure 2. A. 5¢-bromo-substituted pyrrolizidino-2-spiro-3¢oxindole-analog. B. 3,19-(2-bromobenzylidene)-analog.

used occasionally for particular interest of researchers, that is., cholangiocarcinoma cells (KKUM213) were cultivated for bile duct cancer investigation [16]. If possible, the effect on normal cells should be compared so that the desired goal to terminate only the cancer cells can be achieved. In experiments, Androg is used for a baseline to compare with those newly developed. However, the results of Androg on the same cell lines from various experiments sometimes are drastically different; thus standardization and passage of cell lines should be stated, so the results can be compared and correlated among papers. In this review, only active analogs and active cell lines will be described. Apoptosis induction One of the most desirable methods for anticancer drugs to act is via apoptosis or programmed cell death. However, what we have to keep in mind is that test agents should be more specific to the cancer cells than the normal cells, thus at least a normal cell line should be served as a base line for the activity. 3.2

Androg for anticancer According to the data base, Androg acts differently to different cells. Androg was reported to upregulate HLJ1, a tumor suppressor [23], induce autophagic cell death [24], hepatocellular cancer cell cycle arrest and not apoptosis, and by inducing DNA damage [25]. Androg induces cell cycle arrest at G2/M phase and cell death in HepG2 cells (hepatocellular carcinoma) via alteration of reactive oxygen species. At the 3.3

concentration of 40.2 µM, Androg inhibits HepG2 cell proliferation and induces caspase-independent cell death [26]. Benzylidene derivatives of Androg inhibit growth of breast and colon cancer cells in vitro by inducing G 1 arrest and apoptosis [27].

3,19-Benzylidene analogs Benzylidene derivatives were first synthesized by Jada et al. [27], Figure 2B, and the analogs were screened for the growth inhibition/cytotoxicity of the compounds on 60 cell lines, human breast cancer (MCF-7) and HCT-116 were later selected for further investigation for the most effective analogs the 3,19-(2-bromobenzylidene)- (Figure 2B) and 3,19(3-chloro-4-fuorobenzylidene)-analogs gave greater cytotoxic potency and selectivity than Androg. They induced the G1 arrest and apoptosis in both cells. The bromo- analog downregulated cyclin-dependent kinase 4 (CDK4), not the CDK1, in MCF-7. The apoptosis of HCT-166 cells by the two analogs was confirmed by the apoptosis detection kit, annexin V- fluorescein isothiocyanate and propidium iodide [27]. Computer simulation was used to identify docking, binding site identification, in addition to via cell biology studies, it was found that the two analogs directly bind to Ras, block GDP--GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling [29]. 3.3.2


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C. Aromdee

O 14

BzO

14

AcO

O

BnO

11

8

3

4

AcO 19

cytotoxicity against colon and breast cancer cells. The free C-19 carboxylic acid series and the free C3-OH series did not increase much of the cytotoxicity against the HCT-116; IC50 of Androg 19.2 µM on HCT116 [30]. The most effective analogs are shown in Figure 3.

O

11 3

O

8 4

AcO 19

H

O


Figure 3. High potency 19-carboxyl-analogs. A.

B. 14

HO

O 14

HO

O

11 O

8

4

HO O 19

O O

11 3

3

O 19

Si

8

4

HO

H

H

Figure 4. A. 19-O-(triphenylmethyl-) analog. B. 19-TBDPS epoxide analog. TBDPS: Tert-butyldiphenylsilyl.

A.

B. O 14

O

O O

11 O N O O 19

O

11 O

3 H

N

N

N

8 4 H

O N

3

O

O N

Figure 5. A. 3,19-di(1-methylpiperidinyl-4-carboxyl)-analog. B. 3,19-di(pyrazinyl-2-carboxyl)-analog.

This interference of nucleotide exchange is a potential mean to stop the function of oncogenic mutant Ras. Among the halogen substitutions, bromine is the most potent substituent. However, site of the substitution also affects the potency. Halogens are reactive due to its lone pair electrons. 19-Carboxyl analogs The C-19 OH of the Androg (with C-3 and C-14 either substituted or not substituted) was oxidized to carboxylic acid and interacted with methyl- or benzylbromide to get acetyl or benzyl-analogs. The analogs were tested for their 3.3.3

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19-Silyl-substitution Sirion et al. substituted the hydrogen of 19-OH of Androg with three sets of bulky silyl groups, tert-butyldimethylsilyl (TBS), tri-isopropylsilyl- (TIPS) and tert-butyldiphenylsilyl(TBDPS). 12-Hydroxylation, 8,17-epoxidation and substitution with acetyl at the available free OHs were also carried out to the three sets of the bulky 19-silyl analogs [11]. There is no correlation among the non-, mono-, diacetyl of the same 19-silyl series. The silyl substitutions play prominent role on the activity. Without acetyl groups 19-TIPS and 19-CPh3 (Figure 4A) gave the highest activity within the series. The murine leukemia cell line (P388) is most sensitive to all analogs [11]. 3,14-Diacetyl of the TBS series gave potent activity on all cell lines, ED50 of Androg is 15.40 µM on MCF7. All the epoxide series with 19-TBDPS analogs gave similar high activity and also exert topoisomerase II-a inhibition. Topoisomerase II-a cleaves and rejoins double strands of DNA. The epoxide of 19-TBDPS (Figure 4B) was tested on the KKUM213 cells [16], the IC50 on the cell line was 8.0 ± 0.1 µM, whereas 50 µM Androg gave weak cytotoxicity. This analog is a promising candidate as topoisomerase II-a inhibitor as a new anticancer for cholangiocarcinoma, a threatening disease in some parts of Thailand. 3.3.4

H

BnO

O

14-DDA for anticancer Although 14-DDA was not found to exert the cytotoxicity activity on cell lines, A549, BEAS-2B and rat leukemia (KBL)-2H3, modifications of 14-DDA were also carried out. 3.4

3,19-Heterocyclic disubstitution Wei [18] synthesized 17 analogs by substituting the 3-, 19-OHs with either aromatic or aliphatic acids. The 3,19-di (1-methylpiperidinyl-4-carboxyl)- (Figure 5A) is the most effective analog. The activities were determined against four cell lines: adenocarcinomic human alveolar basal epithelial cell (A540), prostate cancer cell lines (DU145), oral carcinoma (KB) and vincristine-resistant KB subline (KB-vin). The GI50 of piperidinyl-analog (Figure 5A) is 1.46 -- 3 µM against the four tumor cells. Whereas the GI50 of the pyrazinyl (Figure 5B) and Androg were 4.87 -- 9.19 and 13 -15 µM, respectively. The size, shape and electronic property of the C-3 and C-19 side chains plays significant role on the activity. 3.4.1

Neoandrographolide for anticancer Neoandrographolide, a glucoside of andrographanin, was hydrolyzed and oxidized to aldehyde and condensed with hexamine to obtain 19-N-hexylamino-8-methylandrographanin (Figure 6A). The analog inhibits tumor growth via blocking 3.5


Andrographolide

A.

B.

C.

O

O O

O O

O

OCH3

H 11

11

3

O 3

3 H

HO

N 19 H

HO

19

OCH3

N

11

AcO

H

19

TBSO

H


Figure 6. A. 19-N-hexylamino-8-methylandrographanin. B. (-)-tetrahydropyran. C. 12-amine-analog.

A.

B.

C. O HO

O

S

O

14

11

11

O

O

S 4

HO O

8

3 4

HO HO

19

H

CH3

19

H

N

H

3

8

3

11

O

O

O O

14

14

OO

8

19

O

S O O

Figure 7. A. 14-lipoyl-Androg. B. 19-sulfonyl-analogs. C. Nicotinoyl-analogs of 14-DDA. 14-DDA: 14-deoxy-11,12-didehydroandrographolide.

neoangiogenesis, and the VEGFR2 causes p38 and ERK1/2 phosphorylation pathway. In vivo activity was also accomplished [31]. 14-DA for anticancer Among the natural analogs of Androg, 14-DA was scarcely investigated. In a modification, the 7,18 double bond was epoxidated, broken and then cyclized to the C-12 to form a tetrahydropyran to obtain (-)-tetrahydropyran (Figure 6B). Other series of the analog were also synthesized by conjugating the 3,19-OHs through an ethyl linkage. None of the synthesized analogs acts against esophageal squamous cell line better than Androg. It’s inhibition activity on a-glucosidase was half of acarbose [32]. Another modification of 14-DA was the substitution with different amines at C-12 on the core structure of 3-acetyl-19-tet-butyldimethylsilyl-14-DA [33]. Six cell lines were employed, P-388 and cultured rat glioma (ASK) cells are sensitive to the derivative. 12-(3,4-Dimethoxyanilinyl)-analog (Figure 6C) gave the highest activity 0.47 -- 5.42 µM, whereas Androg itself exerted the effect with the ED50 of 2.25 and 16.18 µM on the two cell lines. 3.6

4.

Modification for neuroprotective

14-a-Lipoyl-andrographolide (Figure 7A) is a conjugated analog of lipoic acid and Androg. It was claimed to give the potent

activity on anti-HIV, antidiabetic and antibacterial [9]. The compound was tested for inhibiting NF-kB activation in RIN-mb cells [34] and it was 10-fold more potent than Androg. It also exerts protective effects on reactive oxygen species (ROS)-induced RIN-mb cell death for antidiabetes by generating NADPH oxidase-dependent ROS to activate ERK1/2 and AKT1 signaling pathways [35]. Besides that, the analog significantly prevented 1-methyl-4-phenylpyridinium-induced neurotoxicity in SH-SY5Y cells and primary cerebellar granule neurons [36]. This neuroprotective effect is exerted through antioxidation and inhibition of NF-kB activation. Lipoic acid, a nutritional supplement official in USP [1], is a cyclic organosulfur with two sulfur atoms. Sulfur, a member in chalcogen family, possesses 10 oxidation states from -2 to +6. Thus, sulfur can be either oxidized or reduced. To act as an antioxidant, a compound can be oxidized to get rid of the ROS, the compound itself can donate the available proton, or radical species can be formed. Sulfur compounds can be oxidized to sulfide, sulfoxide or sulfone, or can be reduced to sulphydryl or thiol [37]. The active lipoic acid is the R-configuration, (R)-(+)-lipoic acid, it is a cofactor of many enzymes for aerobic metabolism. Lipoic acid is reduced intracellularly to dihydrolipoic acid, this dihydrolipoic acid acts as an antioxidant for ascorbic acid and tocopherol [38]. An antioxidant can be either easier to be oxidized than the substrate or a reducer to encounter with the oxidant.


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C. Aromdee

A.

B. O

O

O

O 11 11 HO O S

O

19

H

H3CO H3CO


3

HO O

3

O

19

H

OCH3

Figure 8. A. 3-hydroxy-19-(2-carbonylthiophene)-12,14-didehydroandrographolide. B. 3-hydroxy-19-(3,4,5-trimethoxxycinnamoyl) 11,12-didehydroandrographolide. A.

B. OH OH

O

O

HO

were tested for the inhibition of the hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and DNA replication. It was found that 3-hydroxy-19-(2-carbonylthiophene) (Figure 8A) is the most effective analog, which inhibits both HBsAg and HBeAg secretions and HBV DNA replication with the selective indices (SI) of 20.3, 125.0 and 104.9. Whereas, the 3-hydroxy-19-(3,4,5-trimethoxy-cinnamoyl)analog (Figure 8B) gave the SI of the inhibition of HBV DNA replication of 165.1 [42]. The structure--activity relationship of the analogs was postulated; the free 3-OH; the conjugated double bonds between C-11 and C-14 or C-12 and C-15; and the heterocyclic aromatic moieties, increase the anti-HBV activity. Androg-succinic acid was esterified with amino acids, glycyl, L-alanyl and L-leucyl [13]. The analogs were used for antiviral and antibacterial infections, antipyretic and antiinflammation [14].

OH

7. O

O

OH

O

OH

Figure 9. A. Warfarin and B. Taxifolin.

5. Anti-dyslipidemic, low-density lipoproteinoxidation and antioxidant activities

Androg was modified by substitution with sulfonyl derivatives at 19-OH. Other modifications are 7,18-epoxide-, 7,18-dihydro, 3-carbonyl-19-aldehyde, 19-(4-methyl-phenylsulfonyl)and 19-(2,4,6-trimethyl-phenylsulfonyl). Only 19-(4-methylphenylsulfonyl) analog (Figure 7B) gave all activities better than Androg [17]. 6.

Antivirals

Androg was modified to 3-(3-nicotinoyl)-14-deoxy,11,12-didehydroandrographolide and used as a core structure for antiHIV synthesis. The 19-OH was modified by esterification with 20 various aromatic or aliphatic acids [39]. It was found that the 19-O-(1-naphthalene acetyl) analog (Figure 7C) exerts a considerably high activity (5.8 µg/ml) with the highest therapeutic index of 34, although the EC50 of Androg is the highest, at 2.78 µg/ml, but the CC50 was 3.64. Uttekar et al. found that 3,19-(3-nitrobenzylidene)-andrographolide and 14-(2¢,6¢-dichloronicotinoyl)-andrographolide exerted in vitro anti-HIV, but the latter gave higher therapeutic index. Both compounds inhibited gp120-mediated cell fusion of HL2/3 cells, molecular target site was identified in both in vitro and computational docking [40]. Esterification of 14-DDA at C-19 with pyridinecarboxylic acid or 2-furoic acid, or 2-thiophenic acid was made for the treatment of hepatitis B virus (HBV) [41,42]. These analogs 1134

Anti-inflammatory

Androg and 14-DDA exhibits anti-inflammatory activity for asthma, through the inhibition of NF-kB [21]. It was found that both compounds inhibited ovalbumin (OVA)-induced increases in total and eosinophil counts, IL-4, IL-5 and IL-13 levels in lavage fluid, and serum OVA-specific IgE level in vivo. 14-DDA was found to block p65 nuclear translocation and DNA-binding activity in the OVA-challenged lung and in TNF-R-stimulated human lung epithelial cells. 14-DDA is less cytotoxic than Androg. Modifications of 14-DDA for treating inflammation and airway disorders have been patented [43].

Interaction of Androg and Andrographis paniculata dried extract with drugs 8.

Herbal medicines are taken without prescription or taken as nutritional supplement, thus it is possible to involve in the effect of drugs taken at the same time. Some herbs strongly interact with the drug-metabolized enzymes. A. paniculata extract is one of the herbal products taken worldwide. Androg and 14-DDA were found to interfere with the drugmetabolizing enzyme UDP-glucuronosyltransferases (UGTs) in Phase II metabolism. They were found to have high specific inhibition toward UGT2B7 [44]. Androg inhibits the CYP1A2 activity, which metabolizes theophylline. Aminophylline is the mixture of theophylline and ethylenediamine. In a pharmacokinetic study in rats, co-administration of Androg and aminophylline was found to have the area under the curve of theophylline in the blood of the subjects significantly higher than administering aminophylline only [45]. However, Chien et al. [46] found that Androg does not affect the metabolism of theophylline but the A. paniculata extract does, which might be due to some other constituents that affect the theophylline metabolism.


Andrographolide

Table 1. Activities and purposes of the patents.


Activities and purposes of the patents

Ref.

Antimicrobial Antiviral agent, anti-inflammatory agent, antibacterial agent, antitumor agent, cardioprotective agent or immunostimulant (phosphoric acid derivative) Extraction for antimicrobial use Inhibition to Candida albicans, and antifungal Antiviral Hepatitis B and its preparations Anti-inflammatory Antitumor agents, anti-inflammation agents, liver protection, anesthetic agent, virus infection, cancer and so on Inflammatory bowel disease Anti-cancer Preparation method antitumor agents Gastric cancer, breast cancer and liver cancer, and has improved therapeutic Farnesoid X Receptor-related diseases: neoplasm cholesterin or blood lipid metabolic abnormality, diabetes, obesity and so on Water-soluble derivative of Androg, its preparation and its applications in medicine Antitumor agents, anti-inflammation agents, liver protection, anesthetic agent Anti-tumor cytotoxic activity BCR--ABL positive lymphoblastic leukemias Androg analogs and its medical application C-15 derivative Natural pharmaceutical compositions for treatment of cancer chemotherapy-induced diarrhea and synergistic treatment of cancer and preparation method thereof Androg compounds, their preparation method and medical application as antipyretic, anti-inflammatory, antiviral or antibacterial agents Miscellaneous Multiple sclerosis Neurodegenerative

and capsules to treat diarrhea induced by cancer chemotherapy and it was found to synergize the treatment of cancer chemotherapy [51]. Androg enhances the therapeutic activity of cisplatin on A2780cisR ovarian cell line [52].

[56]

9.

[57] [58] [59] [41,60,61] [62]

[63] [64] [65] [66]

[5] [67] [68] [69] [61] [51]

[14]

Androg patent update

Most of the patents are the improving method for the preparation of either raw material of Androg, salts and analogs as well as their preparations for therapeutic use. Classification of the patents can be seen in Table 1. 10.

Androg is more cytotoxic than 14-DDA, thus the 14-OH must be involved in the cytotoxicity of Androg. Androg was more modified for anticancer than 14-DDA, whereas, the anti-inflammatory and the antiviral, 14-DDA is preferred. For anticancer, substitution of 19- OH with aromatic, heterocyclic and some silyl derivatives are found to increase the effect. Other modifications were cycloaddition at C-12 and C-13 into two series of oxindoles; cyclization of the 3,19OHs through the substituted benzylidenes. Epoxidation of the double bond at C-7,18 is also included in many series of the developed analogs. For 14-DDA, heterocyclic disubstitution at 3,19-OHs and the substitution at C-12 with aromatic amine, improved the anticancer activity. Size, shape and electronic properties of the modified analogs influence the activity. For anti-inflammation and antiviral purposes, 14-DDA was modified by substitution with aromatic groups at either C-3 and C-19 or C-19 only. Substitution at C-19 OH seems to exert prominent effects in most activities. 11.

[70] [71]

Androg: Andrographolide.

Dried extract of A. paniculata does not affect the pharmacokinetic and pharmacodynamics of warfarin (Figure 9A) in rat [47]. The effect of taxifolin (a flavonoid; Figure 9B) on DU145 cell proliferation was not significant, but it enhanced the effect of Androg by suppressing proliferation and triggering apoptosis in many types of cancer cells by disrupting microtubule dynamics and activating the spindle assembly check point [48]. Androg inactivates androg activates signal transducer and activator of transcription-3 and Akt, which leads to cell apoptosis. It was found to potentiate antitumor activity of gemcitabine in pancreatic cancer cells. The combinative treatment was also tried in vivo [49]. Combination of Androg and 5-fluorouracil was tried to treat colon cancer by sensitizing cancer cells and decreasing the dose of 5-fluorouracil, thus toxicity and side effects due to fluorouracil will be reduced [50]. A. paniculata and Fructus Chebulae mixture is used to prepare tablets

Conclusion

Expert opinion

Androg is well known for a variety of activities: analgesic, antipyretic, anti-inflammatory antibacterial, anticancer, antidyslipidemic; thus after modifications for one activity, analogs are always tried for others, so modifications of Androg for its therapeutic uses will be continuingly explored. A majority of investigations are done for anticancer, antiviral, antiinflammatory agents. However, it is hard to definitely categorize or group the actions of analogs since they possess many activities and diseases can develop into the others, such as inflammation and some viral infections can develop into cancer. Thus, patenting all possible therapeutic activities are included. At present the herb, the herb extract preparations and the injections of pure Androg salts and 14-DDA salts are commercially available [53-55]. The main draw back of Androg is its insolubility in aqueous solvent. To extract Androg from plant as a crude extract, together with other components is easier than to dissolve pure Androg in methanol, which is its most soluble solvent. Making salts from succinate analogs of Androg or 14-DDA to enhance the solubility of the compounds is quite crucial


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since the g-lactone ring is sensitive to strong alkaline, thus only weak alkalis can be used. Weak acids and weak alkalis do not react completely and it is reversible. Removing traces of alkalinity is very important, since it would cause the instability of the drugs. Thus sulfation, which gives stronger acidic properties, is also made so that salt formation and solubility improvement can be achieved with more stability. Therefore, modifications of Androg to increase the aqueous solubility are still going on and being patented. Androg and 14-DDA were most modified since they are abundant in nature; 14-DDA is also easy to transform from Androg. 14-DDA is known to be less toxic than Androg, and was modified for antiviral use more than for anticancer. The substitutions that exert the highest effect on cancer cells are heterocyclic, aromatic. Halogen substitutions of these substituents are more active than others; however, site of the substitution also influences the activity. Nonetheless, some of those with the high potency also exert high toxicity. The heterocyclic formation at C-12 and C-17 via the oxygen, tetrahydropyran, does not enhance any activity of anticancer or a-glucosidase inhibition. Bulky silyl substitutions at C-19, except the TIPS, enhance the anticancer activity. In the activity assessment, Androg is used as a baseline control; however, the activity of Androg on the same cell type varied very much thus result can only be compared within the experiment. The stage at which Androg was found to arrest the cell cycle of the same type of cell is different. Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Standardized methods or passage of the cell lines should be stated. The activity on a normal cell line should be compared, so that specificity to only cancerous cells can be evaluated. Androg is polar on both ends. All of the semi-synthetic analogs might work on their own or via hydrolysis before interaction with their target. Androg had been shown to possess many activities, possibly it can interact with many targets or a biochemical constituents, which might involve in many biological activities. In determining the mode of activity, experimental works for particular activities were designed according to the assumed possible mechanisms. If the result is negative it means that the mechanism is not what had been speculated. But if the outcome is positive it does not mean that the activity is indicative of only the proposed or expected mechanism. Our advanced technologies, laboratories experiment, computer modeling and the outcomes from various means would be compiled and analyzed. Finally, new effective drugs derived from Androg will be achieved.

Declaration of interest The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Affiliation Chantana Aromdee PhD Khon Kaen University, Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, 123 Mitraparp Rd, Muang, Khon Kaen, 40002, Thailand Tel: +66 043 362095; Fax: +66 043 202379; E-mail: [email protected]