General features – Rubia cordifolia (RC) is a medicinal herbaceous perennial flowering plant / vine belonging to Rubiaceae family. It climbs up by getting support from a large tree using tiny hooks grown out of stem and leaves. The name has been offered by the famous Swedish botanist and physician Karl Linnaeus around eighteenth century following his newly invented binomial nomenclature system. RC was originally used as coloring dye for both food and clothes and Ayurvedic medicines. In recent days enormous medicinal and pharmacological applicability of RC has been identified owing to its large reserve of diverse phytochemicals. The herb is commonly known as Indian madder that usually grows around the western Himalayan region or occasionally in other mountainous areas of India. It is also grown in several African nations like in Sudan, Somalia, Southern Angola, South Africa, and Mozambique. In Asian continent besides India, it is seen in Afghanistan, China, Mongolia, Japan, Indonesia, Philippines, and Vietnam. The plant tends to grow at higher elevations of about 9000 feet. The term “cordifolia” in Latin indicates its natural likeness of heart and circulatory system. In ancient times, the roots are still used as a coloring agent. In rural India, the plant root is often used to make red pigment for dying the fabrics. In Bengali, the plant is known as Manjistha (মনজিষ্ঠা) whereas in Hindi it is named as Manjith. Normally, it grows up to the length of ~ 50 – 70 ft. The leaves are evergreen, heart shaped, 2 – 4 inches in length and 0.8 – 1.3 inches wide produced in spirals with 4 – 7 around the central stem. It uses tiny hooks originated at the stems or bottom of leaves. The flowers are small (0.2 – 0.3 inch across the length) having five light yellow petals with dense racemes which appears around June to August trailed by the small berries (0.4 – 0.5-inch diameter) red or black in color. The roots are 4 – 5 ft in length and ~ 0.5-inch diameter in thickness preferring loamy soils with high humus and moderate moisture for the development. In rural areas, the plant is cultivated in arrays for creating fences as boundaries to help keeping away the animals and other live stocks. RC has immense medicinal properties as indicated in ancient Indian Ayurveda. It is a food for the larvae of several varieties of butterflies and moths.
Traditional medicinal uses – RC is often used to treat skin disorders and several cancers. The root extract has astringent taste holding tonic properties. It has antiseptic, antidysenteric and deobstruent properties. It is also effective against rheumatism, ulcers, and inflammations. The plant has antibacterial actions particularly against Staphylococcus aureus. The hydro-alcoholic extract of stems and leaves can act as vermifuge having potent anthelmintic properties. The large reserve of numerous anthraquinone derivatives in RC roots is essentially liable also for antimicrobial, antifungal, analgesic, hypotensive, antimalarial, hypotensive and antileukemic effects. The antimalarial action is due to the quinine content. It is a good food colorant. In Unani and Chinese medicine RC is used to treat dropsy, paralysis, jaundice, amenorrhea, urinary tract obstruction, menstrual disorders particularly heavy bleeding, renal stone, and blood detoxification. The entire plant is a good source of antioxidant. It is also widely known for its veterinary uses for treating maggots, wounds, intestinal worms, liver fluke and dysentery.
Chemical constituents and subsequent physiological effects – The plant contains significant level of alkaloids like quinine, quinones, Benzochromene derivatives, iridoids, triterpenoids and bicyclic hexapeptides. Among the quinones the significant ones are quinine and other anthraquinone derivatives, 1 – hydroxy-2 methoxy anthraquinone, 1, 4 dihydroxy-2 methyl-5 methoxy anthraquinone, 1, 3 – dimethoxy-2 carboxy anthraquinone and rubiadin. The bitter taste of RC is due to its high level of quinine.
Quinine – It is mostly an antimalarial drug which is also effective against Babesia caused by the tick bites. It is common in many rural areas. Besides its beneficial medicinal role the drug has severe adverse effects. In addition to various unfavorable consequences it exerts serious problem producing irregular heartbeat and creating thrombocytopenia (lowering the platelet level).
The compound is extremely toxic particularly to the malaria parasite (Plasmodium falciparum) by virtue of obstructing its hemoglobin metabolizing ability. It inhibits nucleic acid synthesis by affecting to regulate the enzyme purine nucleoside phosphorylase.
Anthraquinones – Majority of the anthraquinone derivatives are physiologically active among those purpurin exists in the highest level. It has anti-genotoxic effect. Similar is the alizarin which has strong anti-genotoxic potency against numerous mutagens as evidenced by the laboratory experiments. Rubiadin another constituent shows strong hepatoprotective effect both in vivo and in vitro experiments. The other anthracene derivatives Rubiasin A, B and C enantiomers show antiproliferative effect particularly on human colon cancer when tested on Col2 cells. Rubicordifolin shows cytotoxicity toward many cancer cells.
Anthraquinone derivatives of Rubia cordifolia
The antiinflammatory role of RC extract of root, stems and leaves is due to its various anthraquinone derivatives. Besides the above effects, they also exert anti-neoplastic or cytotoxic behavior. Approximately seventeen hydroxy quinone derivatives have been isolated and fourteen have been tested for their antioxidant activities. They are the major antioxidant component in RC. The structure activity relation studies indicate that – OH groups on one benzene ring in anthraquinone moiety is absolutely necessary for their antioxidant effect. Its presence particularly at the ortho position greatly increases the activity whereas glycosylation of anthraquinone moiety reduces the effect. The chloroform extract of RC roots shows high level of α – glucosidase inhibition activities which is mostly due to the presence 1,3 dihydroxy-2methyl anthraquinone, 1-hydroxy-2 methyl anthraquinone and 1,2 dihydroxy anthraquinone. The first two compounds show competitive inhibition whereas the last one shows non-competitive inhibition.
Benzochromene or naphtha quinone derivatives – Benzochromeneanalogsare predominantly known to have anticancer, antiinflammatory and neuroprotective effects. In RC, the identified important ones are Mollugin and Rubilactone. Mollugin has several biological properties eg, A) strong anticancer effect by inhibiting TNF – α induced NF – κβ activation, B) Inhibitor of Cholesterol acetyl coenzyme 1 & 2, C) powerful antiinflammatory agent, D) Apoptosis inducer and E) Neuroprotective agent. Almost similar actions are also reported for Rubilactone which is particularly cytotoxic to the hepatoma cells.
Benzochromene derivatives
In addition, furomollugin, Mollugin and Rubilactone show strong suppressive activity on HBsAg secretion from Hep3B cells.
Oleanane triterpenoids – Those are widely known for their antineoplastic actions. The isolated products from RC, Rubiarbonol analogs (A – C) show strong cytotoxic effects toward human cancer cell lines like, HeLa (cervical cancer), BGC-23 (gastric cancer), A549 (lung cancer) and SMMC-7721 (hepatic cancer) (IC50~ 10.75 – 19.87 µg/ml). On the other hand, Rubiarbonol A shows more efficiency on HT-29 (colon cancer) cell lines compared to the others because of – OH at C-28 position. Rubiprasin, Oleanoloic aldehyde acetate from RC also show anticancer and anti-inflammatory effects by preventing eicosanoid synthesis (20-Hydroxyeicosatetraenoic acid). In addition to those oleanolic and ursolic acid are also identified. The other compounds are Oleanolic and ursolic acid. The former has potent anti-inflammatory and antioxidant effects preventing lipid peroxidation as well as suppressing superoxide production. The later one, Ursolic acid provides antitumor effect. It is also a good antioxidant compound and in addition it has the ability to control the high glucose induced apoptosis, possibly by scavenging the reactive oxygen species (ROS). Recent studies further indicate that it enables to control several cancer cells by preventing growth and apoptosis.
These compounds also exert other beneficial effects including the anti-platelet aggregation or blood clotting. Besides these, RC extract also possesses several sterol derivatives like, β – sitosterol, β – sitostenone.
Cyclic hexapeptide – Approximately twenty cyclo-peptides have been isolated from RC. A large section of them have anti-cancer effect. Below is the few bicyclic hexapeptides which show much prominency in that regard. Recently, they are the major focus in the field of cancer research.
Pharmacological actions – The extract of RC root, stem or leaves exert huge pharmacological actions that have many beneficial effects on human health. A few of them are shown below.
Antiinflammatory effect – In laboratory experiments using LPS stimulated macrophage cell line RAW264.7, it has been noticed that RC extract significantly lowers the production of inflammatory mediators like IL-1β, IL-6 and NO generation by controlling the expression of iNOS enzyme. The effective phytocomponents are Physcion, Emodin and 1 – hydroxy-2-methyl anthraquinone and 1 –hydroxy-tectoquinone. Similar results are identified also in case of murine peritoneal macrophages stimulated either by LPS or γ-interferon. Besides iNOS, the expression of COX-2 is also seen to be greatly lowered. In case of in vivo experiments ~ 68 % reduction of edema volume are observed at a dose of 40 mg / Kg body weight.
Neuroprotective effect – The most active component in root extract has been identified to be Mollugin which shows immense neuroprotective effect although there are others within the extract that also indicates several beneficial role in this regard. The effect is mediated by the suppression of pro-inflammatory mediators, upregulation of Heme oxygenase – 1 expression and its activity also accumulation of Nrf-2 along with activation of p38 mitogen activated protein kinase, MAPK pathway. The extract also helps protect progression of Alzheimer’s disease as evidenced in vitro experiment. It inhibits the cytotoxicity due to abnormal accumulation of Aβ within the neuronal cells. In laboratory experiments using animal of Reserpine induced neuronal damage causing orofacial dyskinesia, the extract drastically inhibits tongue protrusions, orofacial bursts, catalepsy and vacuous chewing movements. The concomitant bioassay indicates enhancement of the level of SOD, CAT, GSH and inhibition of LPO and dopamine levels within the forebrain. It is thus logically presumed that the neuroprotective effect could be mediated by the anti-oxidant action.
Immunomodulatory effect – The ethanol extract of RC root or leaves show immunomodulatory effect particularly the protective actions against immunosuppression. The event increases macrophage number, phagocyte index also immunoglobulin level along with the plaque forming cell number. Using animals with peanut-anaphylaxis mouse model it is noticed that RC extract considerably lowers the anaphylactic reactions while lowering the histamine level as well as the IgE production by B-cell line. The protective effect against immunosuppression is further proven in induced animal model using Swiss albino mice are treated with Pb (NO3)2.
Antioxidant effect – A great number of studies have established the powerful antioxidant effect of RC extract which is majorly due to the existence of anthraquinone derivatives. The studies on structural aspect of scavenging ability indicates that – OH group in a phenolic ring of anthraquinone moiety is very necessary to exert the anti-oxidant action. In that regard, – OH group at ortho position exceedingly increases the free radical scavenging effect whereas the glycosylation retards it. The extract considerably lowers lipid peroxidation but enhances the effects of CAT, SOD and GSH which is the indication of antioxidant role.
Antitumor effect – The studies using alcoholic extract shows potent antitumor actions on several cancer cell lines like laryngeal carcinoma (HEp-2), human oral cancer (HN4), human breast cancer cells (SK- BR-3), human non-small cell carcinoma (A-549), leukemia (P-388), human cervical carcinoma (HeLa) and human gastric adenocarcinoma (SGC-790) when tested both in vitro and in vivo animal model. It has been documented that cyclic-hexapeptides isolated from RC has considerable anti-tumor effect exerting strong cytotoxicity when tested on the above tumor cells. But other small molecules in RC also synthesizes numerous anticancer components. For example, Mollugin, napthaquinone can act as a good antitumor compound. It enhances autophagy, inhibits growth and apoptosis. It upregulates mTOR, downregulates FAS gene, activates Nrf-2 with HO-1 using signaling pathways like PI3K/AKT and ERK. It also prevents NF-κβ and its gene dependent products that includes anti-apoptosis factor Bcl-2, Bcl-xl and invasion factors like MMP-9 and ICAM and angiogenesis factor FGF-2 plus VEGF. Several anthraquinones including 1- -hydroxy-2 methyl anthraquinone present in RC also has cytotoxic role on several cancer cell including the malignant skin melanoma, A375 cells.
Effect on blood clotting – The extract of leaves have shown blood clotting activity. It down-regulates thromboxane B2 (TXB2) and upregulates 6-keto-PGF1α level. The extract elevates tissue plasminogen activator (t-PA) activity. The events show its role on the process of blood clotting. The effective anticlotting ingredient has been identified to be Purpurin which is a specific inhibitor (IC50 ~ 6.6 µM) of plasma hylauronan-binding protein (PHBP), a serine protease produced auto-proteolytically enabling to activate Factor VII assisting the extrinsic clotting pathway. Interestingly, Alizarin or Emodin the structural analogs differing from purpurin concerning the position and number of – OH groups are seen to be nonspecific and less active regarding the inhibitory capacity. So, the position / number of – OH imposes selectivity as well as the potency in the overall process.
Anti-HIV action – Human CD4+ T cell line CEM (Child acute lymphoblastic leukemia) tagged with GFP (green fluorescence protein) is infected with HIV-INL4.3 at a multiplicity of infection (MOI) of 0.01 following standard protocol. The infection is monitored by visualizing GFP under microscope. Virus production is analyzed in culture supinate after seven days post infection by p24 antigen capture using ELISA. AZT was used as a standard in the process. RC extract significantly reduced viral infection demonstrating its strong anti-HIV potential.
Toxic effect – Despite its many beneficial role in human health RC has few adverse effects of some of its ingredients particularly if used for longer duration. For example, Rubiadin is proven to be carcinogenic ingredient targeting kidney, liver and large intestine. In addition many of the anthraquinone analogs also show genotoxicity by intercalating within DNA blocking gene expression thereby inducing the cell death. In that regard one should be cautious about the use of RC extracts.