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Abstract

The biological effects of the anthraquinone fraction (AQf) isolated from in vitro cultures of Ophiorrhiza rugosa Wall. var decumbens (Rubiaceae) were evaluated. AQf showed differential activity on reactive oxygen species; it mediated the generation of superoxide radical and inhibited hydroxyl radical and lipid peroxidation. No considerable nitric oxide scavenging activity was observed for AQf. The AQf induced 50% cytotoxicity in Ehrlich ascites carcinoma and Dalton’s lymphoma ascites at concentrations of 130 and 60 µg/mL, respectively. It effectively reduced the inflammation induced by carrageenan in mice. An AQf concentration of 200 mg/kg body weight reduced solid tumor progression in mice. It also prolonged the life span of ascites tumor–bearing mice compared with control mice.

Keywords

anthraquinones antioxidant antitumor anti-inflammatory survival

Introduction

Anthraquinones (AQs) are one of the integral components of the secondary metabolite composition of plants.^1,2 AQs are anthracene derivatives with 2 ketone moieties in the central ring. The functional groups, particularly hydroxyl, attached at specific positions make AQ derivatives widely used anticancer therapeutics.^3,4 Many pharmaceutically important AQ derivatives have been isolated from different plant species. Emodin, aloe-emodin, danthron, and quinizarin are few examples of plant-derived AQs with anticancer activities. These AQs can modulate tyrosine kinase, phosphoinositol 3-kinase, protein kinase C, NF-κB, MAPK, and p53 and p21 signaling cascades, thereby, altering the physiology of cancer cells and induce cytotoxicity.⁵

The genus Ophiorrhiza comprises many medicinally important species such as O rugosa, O mungos, O hayatana, and O pumila. Some of these species have been used in folk medicine as antitussives, expectorants, analgesics, and for the treatment of the amenorrhea and snakebite.⁶ Members of this genus are particularly important for the occurrence of camptothecin, a well-known antitumor compound.^7-9 Many attempts have been made to produce this compound in vitro for a sustainable supply without destruction of natural flora.^9,10 In vitro cultures of O. pumila established by Kitajima et al¹¹ to study the production of camptothecin found that instead of this indole alkaloid, the callus cultures produced large quantities of AQ derivatives. Two new AQs, 1-hydroxy-2-hydroxymethyl-3-methoxyanthraquinone and 2-n-butoxymethyl-1,3 dihydroxyanthraquinone were identified from in vitro cultures of O pumila. According to their study, the camptothecin content from in vitro culture of O pumila was undetectably low, but the production of AQ was high compared with wild type in vivo plants.¹¹ The high content of camptothecin reported from in vitro cultures of Ophiorrhiza by different workers was from differentiated tissues and not from callus or suspension cultures. Very often, undifferentiated callus and suspension cultures fail to accumulate the compounds of interest, whereas shoot and root cultures as well as hairy roots normally produce the same compounds as in the appropriate organs. Irrespective of the undifferentiated or differentiated state, the in vitro cultures of Ophiorrhiza have been found to produce AQs.¹¹

AQs from the family Rubiaceae have been studied for their antimicrobial, hypotensive, and antileukemic activities.^12-14 The anticancer properties and mechanism of action of naturally occurring AQs have been studied in detail.^15,16 The in vitro culture of O rugosa was found to produce large amounts of these pigment molecules in our experiments. The antitumor activity of these AQ compounds from in vitro cultures of O rugosa has not been studied yet. In our study, we isolated and studied the antioxidant, anticancer, and anti-inflammatory properties of anthraquinone fraction (AQf) from tissue cultures of O rugosa.

Materials and Methods

Isolation of AQf From Tissue Cultures of Ophiorrhiza rugosa

In vitro cultures of O rugosa were established in solid Murashige & Skoog medium supplemented with 5% sucrose and different concentrations of benzyl adenine and naphthalene acetic acid¹⁷ and are used for isolating AQs. Briefly, all the plantlets were pooled and freeze dried. The dried plant material was then mixed with 10% aqueous NaHCO₃ solution and extracted with CHCl₃. After evaporation of the solvents, the residue was redissolved in diethyl ether (Et₂O). The ethereal solution was extracted with 1M NaOH. This was then acidified with 1M HCl and extracted with Et₂O.¹⁸ This gave the final extract containing AQs.

Analytical Thin Layer Chromatography of AQf

The AQf are separated with silica gel (60-120 mesh) column chromatography by gradient elution with 0% to 70% methanol (MeOH):CHCl₃. Fractions of 30 mL are collected and evaporated to dryness. The fractions are chromatographed over analytical precoated thin layer chromatography (TLC) plates in the solvent system MeOH:CHCl₃ (10:90 vol/vol) and same fractions were pooled. The presence of AQs was identified by spraying with 10% KOH in ethanol (EtOH). The pooled fraction was then chromatographed by different ratios of CHCl₃:MeOH along with 1 mL of 25% NH₄OH in 100 mL solution. Good separation was found in solvent ratio of 85:14:1(CHCl₃:MeOH:NH₄OH). The colors of 4 bands were identified in visible (VIS), ultraviolet (UV), and in ammonia chamber. The bands were scraped off the preparative TLC plate and desorbed with 0% to 100% CHCl₃:MeOH according to the polarity of the bands. These bands were designated as AQ1, AQ2, AQ3, and AQ4. The UV-VIS absorbance spectra of 4 bands were recorded in an Elico UV-VIS spectrophotometer. The absorbance was taken in EtOH and in alkaline EtOH. The scan range was 200 to 500 nm.

Assays for Antioxidant Activity

The AQf (20 to 200 µg/mL) was studied for superoxide, nitric oxide, and hydroxyl radical scavenging and lipid peroxidation assays. Superoxide radical ( $O_{2}^{-}$ ) scavenging activity was estimated by the riboflavin photoreduction method as described by McCord and Fridovich.¹⁹ Briefly, the reaction mixture contained 3 mg NaCN dissolved in EDTA (6 µM), riboflavin (2 µM), NBT (50 µM), and various concentrations of AQf and phosphate buffer (pH 7.8) in a final volume of 3 mL. The tubes containing the reaction mixture were uniformly illuminated with an incandescent lamp for 15 minutes and the absorbances were measured at 530 nm before and after the illumination. The generation of superoxide radical by AQf was calculated as relative to phosphate buffered saline (PBS) control by the equation

[(Absorbance of AQf - Absorbance of control) / Absorbance of control] \times 100

Hydroxyl radical (OH·) scavenging activity was carried out by the method of Ohkawa et al.²⁰ OH· scavenging activity of AQf was measured by studying the competition between deoxyribose and AQf for the OH· that were generated from Fe³⁺/ascorbate/EDTA/H₂O₂ system (Fenton reaction). The reaction mixture contained deoxyribose (2.8 mM), ferric chloride (0.1 mM), EDTA (0.1 mM), H₂O₂ (1 mM), ascorbate (0.1 mM), KH₂PO₄–KOH (20 mM, pH 7.4), and various concentrations of AQf were incubated for 1 hour at 37°C. The OH· degrades deoxyribose and this is measured as thiobarbituric acid reactive substrate (TBARS).

Nitric oxide (NO) radical scavenging activity was evaluated by Griess reaction.^21,22 Nitric oxide, generated from sodium nitroprusside in aqueous solution at physiological pH, interacts with oxygen to produce nitrite ions. The reaction mixture (3 mL) containing sodium nitroprusside (10 mM) in PBS and AQf (20 to 200 µg/mL) was incubated at 25°C for 150 min. After incubation, 0.5 mL of Griess reagent (1% sulphanilamide, 2% H₃PO₄, and 0.1% naphthylethylene diamine dihydrochloride) was added. The absorbance of the chromophore formed was measured at 546 nm.

In vitro lipid peroxidation assay was determined by TBARS.²⁰ Different concentrations of AQf (20 to 200 µg/mL) were incubated with 0.1 mL rat liver homogenate (25%) containing 30 mM KCl, Tris-HCl buffer (0.04 M, pH 7.0), ascorbic acid (0.06 mM), and ferrous ion (0.16 mM) in a total volume of 0.5 mL for 1 hour. After incubation, 0.4 mL of reaction mixture was treated with 0.2 mL of sodium dodecylsulfate (SDS; 8.1%), 1.5 mL of thiobarbituric acid (0.8%), and 1.5 mL of acetic acid (20%, pH 3.5) for 1 hour in a boiling water bath at 100°C. After 1 hour, the reaction mixture was removed from the water bath, cooled, and 5 mL of pyridine:butanol (15:1) was added, mixed thoroughly, and centrifuged at 3000 rpm for 10 minutes. Absorbance of the clear supernatant was measured at 532 nm against pyridine:butanol. The inhibitions produced by different concentrations of AQf for OH·, NO, and lipid peroxidation were calculated by the equation

[(Absorbance of control - Absorbance of AQf) / Absorbance of control] \times 100

Cells and Animals

Dalton’s lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC) cell lines were propagated in Swiss albino mice by injecting 1 × 10⁶ cells intraperitoneally (IP). The DLA or EAC cells were aspirated from mice, washed with PBS, and diluted to a density of 1 × 10⁶ in PBS. DLA or EAC were then incubated with different concentrations of AQf (40-200 µg/mL) for 2 hours in an incubator to study cytotoxicity. After the incubation, the cytotoxicity of AQf was determined by trypan blue exclusion method.²³

Male Swiss albino mice (20-26 g) were used for the antitumor study. They were purchased from the Small Animal Breeding Station, Mannuthy, Thrissur, India. Six animals were grouped per polypropylene cage and given pelleted feed (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. The experimental protocol was submitted to and approved by the Institutional Animal Ethics Committee (IAEC) prior to commencement of the experiment.

Anti-Inflammatory Activity Carrageenan Induced Acute Inflammation

The animals were separated into 4 groups with 6 animals in each. In animals of each group, acute inflammation was induced by subplanar injection of 20 µL of freshly prepared 1% suspension of carrageenan in normal saline in the right hind paw,²⁴ and paw thickness was measured using Vernier calipers before and at intervals of 1 hour for 4 times after the challenge with carrageenan. Animals were given 2 doses of AQf (100 and 200 mg/kg body weight) half an hour before the carrageenan insult. Diclofenac (DFC) is administered as the standard drug.

Induction of Tumor in Animals

DLA or EAC cells were aspirated from the peritoneal cavity of mice, washed with PBS, and 0.1 mL cell suspension containing 1 × 10⁶ cells was used for injection. Solid tumor was induced on the right/left hind leg of mice by intradermal injection of 1 × 10⁶ DLA cells. Intraperitoneal injection of 1 × 10⁶ EAC cells induced ascites tumor in mice.

Administration of AQf

Solid tumor

The doses of AQf were evaluated by a toxicity study, and safe doses of 100 and 200 mg/kg body weight were determined. Animals were placed into 4 groups containing 6 animals each. The first group of this experiment served as control, which received vehicle alone. Group 2 received cyclophosphamide (15 mg/kg body weight, i.p.) as the standard drug. Groups 3 and 4 received 2 doses of AQf, 100 and 200 mg/kg body weight, respectively, by oral gavage. The drug treatment was started simultaneously with induction of tumor. Calculation of tumor volume started on the seventh day and repeated on every fifth day. The tumor volume was calculated using the formula

V = \frac{4}{3} π r_{1}^{2} \times r_{2} .

In another set of experiments, the treatment was started 10 days after tumor induction, and continued for the following 10 days. Tumor volume was measured starting from the fifth day after tumor induction and repeated at 5-day intervals.

Ascites tumor

Injecting EAC cells into the intraperitoneal cavity of mice produced ascites tumor. The animal grouping and AQf treatment followed the solid tumor model.

Statistical Analysis

The anti/pro-oxidant, cytotoxic, and tumor volume data were analyzed by 1-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. Anti-inflammatory data were analyzed by 2-way ANOVA followed by Bonferroni posttest. The Kaplan–Meier method and log rank test (Mantel–Cox) were used to analyze survival data. Statistical analysis was done using GraphPad Prism Version 5.00 for Windows (GraphPad Software, San Diego, CA).

Results

AQf Contains 4 Distinct AQs

AQf was sequentially resolved by column chromatography and TLC. Analytical TLC showed that AQf contained 4 distinct AQs. Four AQs were obtained by the chromatographic run in CHCl3:MeOH:NH₄OH solution. R_f values were calculated and shown in Table 1. Alcoholic KOH (10%) was sprayed, and it confirmed the presence of AQs. The colors of the bands in daylight, under UV, in NH₃ vapor, spraying alcoholic KOH are noted (Table 2).

Table 1.

R_f of 4 Anthraquinones (AQs) From In Vitro Cultures of Ophiorrhiza rugosa

AQs	R _f
AQ1	0.666
AQ2	0.597
AQ3	0.494
AQ4	0.310

Table 2.

Colors of 4 Anthraquinones (AQs) at Different Conditions

AQs	Daylight	Ultraviolet	NH₃	Alcoholic KOH
AQ1	Bright yellow	Dark	Bright yellow	Red
AQ2	Light yellow	Dark	Fade yellow	Red
AQ3	Light yellow	Dark	Violet	Orange
AQ4	Orange	Dark	Orange	Red

UV spectral analysis showed maximum absorptions between 200 and 300 nm and between 400 and 500 nm. AQ1 showed UV absorptions at 225, 287, and 412 nm. UV absorptions of AQ2 were 212, 240, 310, and 410 nm. The absorptions of AQ3 were 212, 280, and 410 nm. The absorptions of AQ4 were 214, 250, 280, 372, and 410 nm. The UV spectra in alkaline EtOH showed characteristic bathochromic shift of AQs in the absorption (Figure 1).

Figure 1.

Ultraviolet (UV) spectra of 4 anthraquinones isolated from Ophiorrhiza rugosa

AQf Differentially Scavenges Oxidative Radicals

Free radicals are electron-deficient reactive atoms or molecules that can damage normal and biologically important macromolecules such as lipids, proteins, and DNA. Here we evaluated the efficiency of AQf in scavenging different free radicals and its preventive effect on lipid peroxidation. AQf inhibited OH· radicals in a concentration-dependent manner. The IC₅₀ for OH· radical scavenging was 190 µg/mL (Figure 2A). But in contrast, $O_{2}^{-}$ radicals were generated by the AQf, which was clear from the oxidation of nitroblue tetrazolium (NBT) molecules (Figure 2B). NBT molecules on oxidation with $O_{2}^{-}$ radicals are converted to blue colored diformazan. The intensity of color of diformazan is proportional to the concentration of $O_{2}^{-}$ generated. Here we found that AQf-treated groups contain more oxidized NBT than control groups. It suggests that AQf was generating $O_{2}^{-}$ rather than scavenging them. No biologically significant NO radical scavenging activity was found for AQf (Figure 2C). Lipid peroxidation was effectively inhibited by AQf in a concentration-dependent manner (Figure 2D).

Figure 2.

Anthraquinone fraction (AQf) exhibits both antioxidant and pro-oxidant activity

AQf Induces Death in EAC and DLA Cell Lines

Anthraquinone fraction induced 50% cell death in EAC cells at a concentration of 60 µg/mL (Figure 3A). In DLA cell lines, AQf brought about the same effect at a concentration of 130 µg/mL (Figure 3B).

Figure 3.

Anthraquinone fraction (AQf) exhibits cytotoxicity in cancer cell lines

AQf Inhibits the Acute Inflammatory Effect of Carrageenan

Carrageenan is a sulfated polysaccharide that induces acute inflammation and paw edema when injected interdermally. AQf treatment 30 minutes prior to carrageenan insult reduced the edema formation in the hind paw. At the first hour, DFC and 2 concentrations of AQf (100 and 200 mg/kg body weight) did not show any significant reduction of paw edema. In the DFC-treated group, at the second hour significant reduction of edema was noticed, whereas there was no such reduction in the AQf-treated groups. At third hour and fourth hour, significant reduction of edema was noticed for the AQf treated groups (Figure 4). Both the concentrations of AQf were found to have similar potential to reduce the edema volume.

Figure 4.

Anthraquinone fraction (AQf) inhibits the acute inflammatory effect of carrageenan in mice

AQf Prevents and Regresses DLA-Induced Solid Tumor

Two concentrations of AQf were studied for their antitumor activity. In the simultaneous treatment group, there was an increase of tumor volume until the 17th day posttumor inoculation. In the subsequent days, a reduction in the tumor volume in the animals treated with AQf was observed. Tumor growth was effectively suppressed by AQf treatment. Compared with control and CTX-treated groups, the tumor volumes were less for AQf-treated groups on the seventh day (Figure 5A). In the animal group, where AQf treatment was started after successful establishment of solid tumor in the limb, the progression of increase in the tumor volume was less severe compared with the control group (Figure 5B). These data indicated that AQs in the AQf have effective antitumor activity.

Figure 5.

Anthraquinone fraction (AQf) exhibits antitumor activity by reducing tumor burden in mice. Two concentrations of AQf (100 and 200 mg/kg body weight) were given as oral gavage either simultaneously (A) with tumor induction or 10 days posttumor development (B). ***P < .001 control versus AQf (200 mg/kg body weight) on the 22nd and 27th days for (A) and from 15th day for (B).

AQf Increases Life Span of Ascites Tumor–Bearing Animals

Two concentrations of AQf were tested for their efficacy to delay the death of ascites tumor–bearing mice. The median days of survival of the control group were 15.5 and 16 days in simultaneous and developed treatment models, respectively. Median survival in animals treated with 200 mg/kg body weight of AQf was 28 and 25.5 days in the 2 treatment models. The increases in the life span of AQf-treated mice were comparable with CTX-treated group (Figures 6A and 6B).

Figure 6.

Anthraquinone fraction (AQf) increases life span of ascites-bearing mice

Discussion

We studied the antioxidant, antitumor, and anti-inflammatory activities of crude AQf from in vitro cultures of O rugosa. The data emphasize the presence of active AQs in the crude mixture of AQf showing the antitumor and anti-inflammatory activities. AQs are phenolic compounds showing a wide range of pharmacological properties, which are the basis for different applications in the broad area of pharmacy and medicine.^25,26 AQs are known to have antioxidant activity, but it is also reported that the cancer cell toxicity of many AQs is due to the generation of ROS. In our study, we tested crude fraction of AQs for antioxidant and cytotoxic potential. The antioxidant assays showed interesting results of differential regulation of ROS; on one hand AQf mediated the generation of superoxide radical, but on the other hand, it scavenged hydroxyl radicals. Although we found a statistically significant reduction in NO radical production, the highest concentration of AQf could scavenge only 15% of NO radicals. AQs can accept electrons and form semiquinone free radicals that can transfer electron to dioxygen and convert them to superoxide radicals. Despite the generation of superoxide radicals, AQf was able to inhibit 60% of OH· radicals, and prevent lipid peroxidation. In the hydroxyl radical assay system, the OH· radicals are generated via the Fenton reaction. AQs are known to chelate metal ions. The inhibitory effect on the generation of OH· radical by AQf might be caused by the chelation of Fe⁺⁺ ions in the reaction mixture and suppression of the Fenton reaction. Lipid peroxidation depends on hydroxyl radical generation.²⁷ AQf showed significant reduction in lipid peroxidation. The suppression in the generation of hydroxyl radical seems to be associated with the reduction of lipid peroxidation.

The cytotoxic efficacy of AQf was studied on EAC and DLA cells. AQf showed an LD₅₀ at concentrations of 130 and 60 µg/mL in EAC and DLA, respectively. The cytotoxic activity of many AQ derivatives has been previously documented.^15,28 One of the major mechanisms of action of AQ cytotoxicity is ROS mediated.²⁹ The generation of superoxide was observed for AQf in the in vitro assays. Previous workers have shown that electron paramagnetic resonance and electrochemical studies of AQ such as emodin could be easily reduced to semiquinone form and in the presence of oxygen and the semiquinones generate ROS, mainly superoxide.³⁰ Intracellular increase in ROS often leads to apoptosis, which is considered to be the mechanism of action of cytotoxicity in cancer cells mediated by AQs.³¹

Anti-inflammatory activity was observed in AQf-treated groups. Inflammation is a complex, difficult to control, and self-perpetuating process that is responsible for development of diseases such as atherosclerosis and cancer.³² Little work has been done on the anti-inflammatory activities of AQs. Carrageenan induced interdermal inflammation, which is involved with both nonphagocytic and phagocytic inflammatory responses. Leukocyte infiltration and mast cell damage occurs in this event.³³ The exact mode of inhibitory action on inflammation by AQ is not known at this point in time. The inhibition of lipid peroxidation and the OH· radical scavenging may be correlated with the anti-inflammatory action of AQf.

The cytotoxic activity in DLA and EAC cells prompted us to study the antitumor activity of AQf in vivo. The antitumor activity was evaluated in 2 model systems; solid tumor and ascites tumor. AQf treatment reduced the solid tumor burden very effectively. In the simultaneous treatment experiment, a good response was observed as a reduction in the tumor volume. In the developed tumor experiment, AQf treatment was started 10 days after tumor induction; only 200 mg/kg body weight AQf resulted in marked decrease in tumor volume. Treatment with AQf increased the life span of ascites tumor–bearing animals. The antitumor activities of AQs have been well documented in the literature.^34,35 The antitumor activities of AQs are associated with the side group substitutions they possess.³⁶ Different side groups contribute to varying degrees of cytotoxic and antitumor potential for the compound. Some substitutions will render them capable of producing ROS and others help intercalate to DNA and subsequently block the stabilization of the cleavable complex between TopII and DNA.³¹

In conclusion, it is obvious that the AQs in the AQf have potential to induce death of cancer cells, inhibit inflammation, and reduce tumor burden. Activity-based fractionation is needed to isolate and identify the active ingredient/s in the AQf.

Footnotes

Acknowledgements

We thank Matthew E. Sweeney for careful reading of the article.

The author(s) declared no potential conflicts of interests with respect to the authorship and/or publication of this article.

The author(s) received no financial support for the research and/or authorship of this article.

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Antitumor Activities of an Anthraquinone Fraction Isolated From In Vitro Cultures of Ophiorrhiza rugosa var decumbens