Sub-Saharan Rubiaceae: A Review of Their Traditional Uses, Phytochemistry and Biological Activities

Rubiaceae family is a large family of 630 genera and about 13000 species found worldwide, especially in tropical and warm regions. These plants are not only ornamental but they are also used in African folk medicine to treat several diseases. Based on online published data and library bibliographic research, we herein reported accumulated information related to their traditional usages in sub-Saharan traditional medicine, their chemical composition and the screened pharmacological activities. Indeed, more than 60 species are used for more than 70 medicinal indications including malaria, hepatitis, eczema, oedema, cough, hypertension, diabetes and sexual weakness. Through biological screening following leads supplied with traditional healers, many of these plants exhibited antimalarial, antimicrobial, antihypertension, antidiabetic, antioxidant and anti-inflammatory activities. Bioactive compounds including indole alkaloids, terpenoids and anthraquinones have been isolated from these bioguided fractionation studies. It is evidence that great attention has been paid to species such as Nauclea latifolia, Morinda lucida, Mitragyna inermis and Crossopteryx febrifuga; however, several compounds should be waiting to be discovered since none of these plants has been systematically investigated for its biochemical composition. According the current global health context with the recrudescence of HIV, much effort should be oriented towards this virus when screening Rubiaceae.

INTRODUCTION

The use of plant-based systems continues to play a key role in health care. Many reports estimated that approximately 80% of the population in developing countries still relies on Traditional Medicine (TM) for their primarily health care (WHO, 2011; Hostettman and Marston, 2002). In some African countries such as Ghana, Mali, Nigeria and Zambia, the first line of treatment for 60% of the children with high fevers, resulting from malaria, is the use of herbal medicines at home (WHO, 2003). In these societies, the tradition of collecting, processing and applying plants and plant-based medications have been handed down from generation to generation. Traditional medicine, with medicinal plants as their most important component are sold in marketplaces or prescribed by traditional healers in their homes (Von Maydell, 1996).

The development of resistance to most of the available antimicrobial agents and the high costs of treatment consequent upon this resistance has necessitated a search for new, safe, efficient and effective agents for the management of infections (Okwu and Uchenna, 2009). This research for new effective agents against infectious diseases and other diseases such as, cancers, diabetes, cardio-vascular, neurological, respiratory disorders, etc has led to increased interest in existing information about the remedies of these diseases from natural sources, principally the plants (Karou et al., 2007; Ouattara et al., 2007). Because of this strong dependence on plants as medicines, ethnopharmacological studies have been conducted to determine their safety and their efficiency and on the other hand to find out new active principles from plants (Zongo et al., 2010; Ouattara et al., 2011a,b).

Rubiaceae are among plants of wide usage in traditional medicine that are continuously screened in laboratory for their pharmacological properties. According to their wide distribution, these plants are used in all parts of the world as ornamentals, foods and remedies. The most economically important members of the family are the two species of shrub Coffea canephora (also known as Coffea robusta) and Coffea arabica, used in the production of coffee. Gardenia jasminoides is a widely grown garden plant and flower in frost-free climates worldwide. Several other species from the genus are also seen in horticulture. The genus Ixora also contains plants seen cultivated in warmer climate gardens.

In medicine, trees of the genus Cinchona are of great interest because of their alkaloids, the most familiar being quinine, the first effective agent in treating malaria. In this last topic the Rubiaceae family received great attention by scientists. The present review is focussed on the Rubiaceae growing in Sub-Saharan Africa. The online published studies enable us to summarize the uses in indigenous TM and the compounds occurring in theses plants. This research finally discussed the biological activities exhibited by theses plants.

Botanical data: Rubiaceae is a family of flowering plants, variously called the madder family, bedstraw family or coffee family. The family takes its name from the Madder genus Rubia. Other plants such as Gardenia, Cinchona, Gambier, Ixora, Naucleaceae and Theligonaceae have been included in the family. Now-a-days there are about 630 genera and more than 13000 species in the family; making the Rubiaceae one of the six largest angiosperm families including Asteraceae, Orchidaceae, Fabaceae, Poaceae and Euphorbiaceae in terms of number of genera and species. Rubiaceae species are concentrated in warmer and tropical climates around the world (Dalziel, 1957). A wide variety of growth forms are present in the Rubiaceae. Shrubs are most common, but members of the family can also be trees, lianas or herbs. Species are mainly woody, less than 20% of the genera are herbaceous. A large number grow in ub Saharan Africa. The most represented are Anthospermeae, Morindeae, Spermacoceae, Cinchoneae, Naucleae, Coffeae, Gardenieae and Pavetteae.

Traditional uses of sub-Saharan Rubiaceae
Rubiaceae members used in Sub-Saharan traditional medicine: There are more than 30 online publications on Sub-Saharan ethnobotany. The main studies are published in Journal of Ethnopharmacology, Journal of Ethnobiology and Ethnomedicine, African Journal of Biotechnology and African Journal of Traditional, Complementary and Alternative Medicine. A total 73 Rubiaceae species all growing in sub tropical Africa and distributed into 34 genera are documented as having medicinal value in this part of the world. These genera are Anthospermum, Borreria, Breonadia, Canthium, Chassalia, Cinchona, Coffea, Craterispermum, Crossopteryx, Diodia, Fadogia, Feretia, Galium, Gardenia, Hallea, Keetia, Macrosphyra, Mitracarpus, Mitragyna, Morinda, Mussaenda, Nauclea, Oxyanthus, Oldenlandia, Pausinystalia, Pavetta, Pentas, Psychotria, Rothmannia, Rubia, Rytigynia, Sarcosaphelus, Spermacoce, Uncaria and Vangueria (Fig. 1). The genus Pentas with the following species: Pentas bussei K. Krause, Pentas decora, Pentas hindsioides, Pentas lanceolata, (Forssk.) Deflers, Pentas lanceolata (Forssk.) Defl. Subsp. quartiniana (A. Rich.) Verdc., Pentas longiflora Oliver, Pentas micrantha, Pentas purpurea, Pentas shimperana subsp. occidentalis (Hook.f.) Verde., Pentas schimperiana (A. Rich)Vatke, Pentas zanzibarica (Klotsch) Vatke; is the most represented followed by the genus Gardenia and Canthium with the species Gardenia aqualla Stapl and Hutch, Gardenia cornuta, Gardenia erubescens Stapl and Hutch, Gardenia imperilalis, Gardenia sokotensis Hutch and Kew Bull, Gardenia ternifolia and Gardenia triangacantha. Canthium glaucum Hiern., Canthium multiflorum Schum and Thonn, Canthium oligocarpum Hiern, Canthium setosum Hiern., Canthium vulgare Bullock, Canthium zanzibarica Klotzsch. and Canthium spp; respectively. The present list of Sub-Saharan medicinal Rubiaceae could not be exhaustive since the data are based on the internet and library bibliography research. In this study there is a disparity of ethnobotanical data published online. Some countries such as Ethiopia, Nigeria, Cameroon and Kenya have more than 10 online publications on ethnobotanical studies, while these data are missing for other countries such as Togo, Niger and Benin. Of course still very little is known about the medicinal practises and plants used in the folk medicine of these countries.

Concoction and mode of administration: According to published ethnobotanical data, Rubiaceae plant parts used for medical preparations are leaves, bark, roots and fruits. In some cases the whole plant is used including the roots. The most frequently used plant parts are the leaves followed by the bark, stem and roots. Single plants may be used alone or in association with other plants or with other material of animal or mineral origin. Remedies are mainly prepared in the form of powder, concoction and decoction. The methods of administration of herbal medicines are internal, particularly by oral absorption and external: poultice/topical application or bathing.

Ailments: The remedies are used in the management of many diseases including abdominal irritation, abortion, abscesses, anaemia, arthritis, ascariadis, ascite, asthenia, baby growth delay, chancre, chicken pox, conjunctivitis, constipation, cough, cryptococcal meningitis, dermatitis, diabetes, diarrhoea, dizziness, dysentery, dysmenorrhoea, eczema, epilepsy, evil eye, evil spirit, fever, filariasis, gastritis, general weakness, gonorrhea, headache, hemorrhage, hepatitis B, hydrocele, hypertension, itchy rashes, infant umbilical pains, internal inflammation, jaundice, kidney diseases, leprous macular, lumbago, lymphadenitis, madness, malaria, mental disorders, measles, mycoses, obesity, oedema, ovarian cyst, paralysis and nerve diseases, pinworm, poisoning, pubic lice, respiratory infection, rheumatism, ringworm, scabies, sexual impotence, snake bites, splenomegaly, sterility, syphilis, threatened, tapeworm, trypanosomiasis, urinary retention, urinary tract infection, vomiting and wounds (Table 1).

Fig. 1:	Rubiaceae members used in Sub Saharan Africa for medicinal purposes

Malaria and microbial infections are the main diseases cited. Overall, the cited diseases cover the main of indigenous diseases; indeed, Rubiaceae may be considered as a major component of sub-Saharan folk medicine.

Chemistry of Rubiaceae: Various natural products occur in Rubiaceae plants. Extensive phytochemical investigation has been realized regarding the natural occurrence of terpenoids, anthraquinones and indole alkaloids in the family. The occurrence of alkaloids seems to be a rule in this family, although Leal and Elisabetsky, (1996) demonstrated the absence of alkaloids in Psychotria carthagenensis. The alkaloids of Rubiaceae are indolique alkaloids. They may occur in tetracyclic or pentacyclic rings (Fig. 2). The occurrence of alkaloids in some Rubiaceae is well documented. The leaves of M. inermis contain tetracyclic and pentacyclic oxindole and indole alkaloids including uncarine D, rhynchophylline, isorhynchophylline, rotundifoline, isorotundifoline, ciliaphylline, speciogynine, pteropodine, uncarine F, mitraphylline, isomitraphylline and mitraciliantine (Toure et al., 1996). The proportion of these compounds is variable and depends on the growing location and the season of harvest. A number of monoterpene indole alkaloids including nauclefine 1 and 2 have been isolated from Nauclea species. The main alkaloids of N. pobeguinii were identified as strictosamide, carboxystritosidine and methylangustoline (Fig. 3). N. latifolia contains diverse phytochemicals such as alkaloids, flavonoids, steroids and glycosides. Earlier workers on the plant isolated a series of alkaloids from it. Naucleafoline, nauclechine and naufoline were isolated from the leaves. Other alkaloids isolated from the plant include naucletine, nauclefine, naucledidinal and epinaucleidinal, augustine and card-ambine (Hotellier et al., 1975, 1979). Naucleidal and epinaucleidal (Fig. 4) have been isolated from an antiviral preparation produced by roasting Nauclea latifolia fruits (Morah, 1994); furthermore, five monoterpene indole alkaloids, naucleamides A to E (Fig. 4), were found to occur in the bark and wood of the plant. Naucleamide E was the unique monoterpene indole alkaloid possessing a pentacyclic ring system with an amino acetal bridge (Shigemori et al., 2003). From the stem bark of Mitragyna africanus collected in Nigeria, seven Corynanthetype oxindole alkaloids, i.e., rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, ciliaphylline, rhynchociline and isospecionoxeine, were isolated. Furthermore, a new indole alkaloid 9-methoxy-3- epi-α-yohimbine (Fig. 4) was isolated as a minor component (Takayama et al., 2004). However, crossopterine was found to be the main alkaloid occurring in the bark of Crossopteryx febrifuga (Tona et al., 2000). Chemical compounds isolated from different parts of the plant also include quercetin and non-quercetin containing flavonoids from the leaves and bisdesmonic saponins and triterpene saponin from the stem bark. Similarly, from the bark of Mitragyna inermis, Cheng et al. (2002) isolated two 27-nor-triterpenoid glycosides, named inermiside I and II (Fig. 2). A detailed phytochemical study of Pentas longiflora resulted in the isolation of compounds belonging to chemical families including naphthoquinones, anthraquinones, coumarins and steroids (Fig. 5) (El-Hady et al., 2002).

Fig. 2:	Chemical structure of inodle alkaloids and terpenoids occuring in Rubiaceae

Fig. 3:	Chemical structure of compounds isolated from N. pobeguinii

Similarly the investigation of P. bussei resulted in the isolation and identification of compounds presented in Fig. 6. These were new highly oxygenated naphthohydroquinones e. g., methyl 8-hydroxy-1,4,6,7-tetramethoxy-2-naphthoate methyl 1,8-dihydroxy-4, 6, 7-trimethoxy-2-naphthoate; New naphthohydroquinones of the benzochromene type e.g., methyl 5,10-dihydroxy-7-methoxy-3-methyl-3-(4-methyl-3-pentenyl)-3H-benzo[f]chromene-9-carboxylate methyl 5,10-dihydroxy-7-methoxy-1,1,3atrimethyl-1a,2,3,3a,10c,10d-hexahydro-1H-4-oxacyclobuta[3,4]indeno[5,6-a]naphthalene-9-carboxylate 9-methoxy-2,2-dimethyl-2H-benzo[h]chromene-7,10-diol, 9-methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-benzo[h]chromene-7,10-diol and 7-hydroxy-3,3-dimethyl-10-methoxy-3H-benzo[f]chromene-8-carboxylic acid.

Table 1:	Medicinal indications of sub-Saharan Rubiaceae
NA: Non available data

Further more the already known α-stigmasterol was isolated from the roots of the plant (Bukuru et al., 2002). The same study conducted on P. parvifolia revealed that the chemistry of the plant was mainly similar to that of P. bussei (Bukuru, 2003).

Screened biological activities of Rubiaceae plants: Rubiaceae species have been screened for various biological activities both in vitro and in vivo using animal models. The main biological activities were antiplamodial, antibacterial, antiinflammatory and antidiabetic activities.

Antiplasmodial activities: The antimalarial activity is the biological property that received great attention by scientists interested in Rubiaceae investigation.

Fig. 4:	Chemical structure of compounds isolated from N. latifolia

Fig. 5:	Chemical structure of compounds isolated from Pentas longiflora

Extracts were tested on parasite cultures in vitro to check if they affect the viability of the main malaria parasites, Plasmodium falciparum (Pf). Parasites are often fresh clinical isolates obtained from untreated malaria patients or reference Chloroquine-Sensible Plasmodium falciparum (CQSPf) such as strain D6 or Chloroquine-Resistant Plasmodium falciparum (CQRPf) strains such as 3D7.

Fig. 6:	Chemical structure of compounds isolated from Pentas bussei

Parasites are grown as described by Trager and Jensen, (1976). The quantitative assessment of antimalarial activity in vitro is determined by means of the radioisotope technique with incorporation of [3H] hypoxanthine based on the method described by Desjardins et al. (1979) and Schulze et al. (1997). A light microscopy technique using giemsa-stained smears and colorimetric method that includes 3-acetylpyridine as a substrate for malaria parasite lactate dehydrogenase has been used with the advantage that radio labelled substrates are not required has been developed (Karou et al., 2003; Makler et al., 1993).

A large number Rubiaceae species that are used in TM have been tested in vitro for the antimalarial activities. Two mains reasons prompted theses studies. Firstly, Sub-Saharan Africa possesses many endemic malarious regions and surly the indigenous people have a long experience in antimalarial plant usage. Secondly, since the main antimalarial drug quinine is of Rubiaceae origin, the researchers suppose that similar compounds with similar properties may occur in the family.

Many Rubiaceae crude extracts have been tested with success on P. falciparum. Table 2 displays several results recorded with Rubiaceae crude extracts. The antimalarial activity is expressed in terms of IC_50, drug concentration causing 50% death of the initial parasite amount. Indeed, (Benoit-Vical et al., 1998) fond IC₅₀ of 0.6 μg mL^-1 using aqueous extract of Nauclea latifolia on the Columbian multidrug Resistant Pf CQRPf FcB1, similar IC₅₀ value was recorded on Nigerian clinical isolate by Menan et al. (2006). However, the ethanol extract of the same plant yielded IC₅₀ of 8.9 μg mL^-1 on the same Columbian CQRPf strain (Zirihi et al., 2005) (Table 2). Globally IC₅₀ values below 5 μg mL^-1 were recorded with chloroform or methylene chloride extracts both on MDRPf or CQSPf or clinical isolates. With Pavetta crassipes, chloroform extract yielded 1.02 and 1.23 μg mL^-1 on Pf D6 and W2, respectively (Sanon et al., 2003a); 4.36 and 4.82 μg mL^-1 with Mitragyna inermis on Pf W2 and 3D7 respectively (Traore-Keita et al., 2000). Methylene chloride extract of Canthium setosum yielded 2.77 and 4.8 μg mL^-1 on Pf (3D7 and K1, respectively while the methanol extract of the same plant yielded IC₅₀ up to 6 μg mL^-1 on the same strains (Weniger et al., 2004). Thus chloroform and methylene chloride appeared to be the best solvents for Rubiaceae antimalarial agent extraction. According to the recorded IC₅₀, N. latifolia is the most efficient antimalarial Rubiaceae. Although, interesting IC₅₀ values of crude extracts were recorded with other species such as Enantia pylocarpa Oliver (Annonaceae) and Croton lobatus L. (Euphorbiacea), Rubiaceae are considered as the main source of antimalarial drugs (Atindehou et al., 2004).

The main chemical group responsible for their antimalarial activity was identified as alkaloids. Indeed alkaloids of P. crassipes, N. latifolia and M. inermis were tested with success for antiplasmodial activity. Moreover, Ancolio et al. (2002) found synergistic effect of the combination of total alkaloids of M. inermis and N. latifolia on Pf D6 and additional effect on Pf W2. The activity of Uncarine (Fig. 2) the main alkaloid of M. inermis on the CQRPf strain W2 was not correlated with its concentration in the leaves of the plant (Fiot et al., 2005). Mesia et al. (2010) investigated for the antimalarial activity of Nauclea pobeguinii. Five compounds were isolated in this study: (5S)-5-carboxystrictosidine, 19-O-methylangustoline, 3-O-β-fucosyl-quinovic acid, 3-ketoquinovic acid and strictosamide (Fig. 4). Only 19-O-methylangustoline showed a moderate antiplasmodial activity (IC₅₀ = 26.5 μg mL^-1) on a Ghana clinical Pf, the other compounds were devoid of antimalarial activity.

Globally Rubiaceae extracts remain active on the clinical isolates according to few screening that used theses strains. This is benefit for indigenous people since they are the effective pathogens causing the disease in the concerned zone, in contrast with the reference strains that may have been isolated in other region, thus not reflecting the reality.

The extracts are now being test on animal model for the antiplasmodial activity. Ethanolic extracts of the stem bark of Crossopteryx febrifuga was investigated against early, residual and established malaria infections in vivo using Swiss albino. Results revealed that an amount of 200 mg kg^-1 per day appeared to be the effective therapeutic dose for the animals. Indeed, Salawu et al. (2008) noted that the administration of 100 mg kg^-1 of methanolic extract account for 84.7% reduction of parasitemia versus 76.6% for 5 mg kg^-1 chloroquine phosphate. More recently Mesia et al. (2010) found that the aqueous and 80% ethanol extract of N. pobeguinii displayed moderate in vitro activity with IC₅₀ values of 44 and 32 μg mL^-1, respectively. Daily oral dosing of the extract, containing 5.6% strictosamide, at 300 mg kg^-1 resulted in 86% reduction of parasitaemia in the 4-day Plasmodium berghei mouse model and 75% reduction in the Plasmodium yoelii N67 model. Prolonging oral dosing to 2x5 days, with an interval of 2 days and oral administration induced 92% reduction of parasitaemia and a mean survival time of 17 days. Strictosamide, the putative active constituent, may be metabolically activated in the gastrointestinal tract after oral administration.

Antibacterial activity: The crude extracts of the Rubiaceae and their subsequent partitioning gave fractions exhibited a broad spectrum antibacterial activity on several microbial pathogens including reference strains and clinical isolates. Indeed, reference strains such as Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 are often used. The extracts are tested using the agar diffusion assay or the broth microdilution assay. In the first case the antimicrobial activity is expressed as inhibition zone diameter around the disk or the well. Using this assay, (Adomi, 2008) screened the aqueous extract of Morinda lucida. The recorded inhibition zone diameters with 1000 mg mL^-1 extract varied from 14 to 25 mm with gram positive and gram negative bacteria (Table 3). The agar diffusion assay is efficient for the quantification of the antimicrobial activity; however, the solubility and the diffusion of some extract in the agar medium can be a limiting factor. Hence, some authors prefer the microdilution assay rather than the agar diffusion. The microdilution allows expressing the activity in term of drug concentration killing microorganisms. The Minimal Inhibitory Concentration (MIC) is then recorded as lowest extract concentration demonstrating no visible growth in the broth and the Minimal Bactericidal Concentration (MBC) as a lowest extract concentration killing 99.9% of bacterial inocula. MBC/MIC ratios can be calculated to appreciate the effect of the extract on the tested microorganisms; indeed MBC/MIC ratios greater than 1 indicate microbiostatic effect of extract, while ratios under 1 indicate microbicide effects of extracts (Karou et al., 2005).

Using the broth microdilution assay, Asase et al. (2008) found good inhibition of both gram positive and gram negative bacteria by the acetone extract of Mitragyna inermis rather than the n-hexane extract. Okoli and Iroegbu (2004) combined the microdilution and the agar diffusion to screen the antibacterial activity of ethanolic and aqueous extracts of N. latifolia. Four references strains, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6051, Pseudomonas aeruginosa ATCC 10145 and Escherichia coli ATCC 117755; in addition with clinical isolates of S. aureus and E. coli were tested. The ethanol extract was found to be more active on the tested microorganisms. In the agar diffusion assay the inhibition zone diameters recorded ranged from 6.60 to 17 mm while MIC varied from 12.50 to up to 50 mg mL^-1 in microdilution assay (Table 3). Further, the extract was found to be bacteriostatic to both Gram positive and Gram negative strains. Working on the same plant for the same purposes, Okwori et al. (2008) found that the alcoholic and aqueous extracts exhibited both bactericidal and bacteriostatic activities on gram positive bacteria while the gram negative ones seemed to resist to the extract. Another way to appreciate the antimicrobial activity is the time-kill assay.

Table 2:	Antimalarial activities of sub-Saharan Rubiaceae
*: Clinical isolates of Plasmodium falciparum; Nt: Not tested; -: Non available data

This allows monitoring the decrease of bacterial amount as a function of the time. The assay consists of exposing bacteria to a drug concentration greater that the MIC and to perform cell enumeration at regular time interval. This assay was used by Akomo et al. (2009) who demonstrated that no viable microorganism remained in the medium for Enterococcus faecalis CIP 105150 and Escherichia coli CIP 105182 after 9 h and 11 h exposition to 1.25 and 2.50 mg mL^-1 methanol extract of Canthium multiflorum respectively. Similarly, Zongo et al. (2009) found that 9 h exposition to 3 mg mL^-1 alkaloids of M. inermis killed the total inoculums.

According to results recorded with Rubiaceae, main authors found greatest activity of ethanol extracts, showing the strong capacity of this solvent to extract the antibacterial compounds of Rubiaceae.

Table 3:	Antimicrobial activities of sub-Saharan Rubiaceae
Data are the value of minimal inhibitory concentration for broth microdilution assay and values of inhibition zone diameter in for agar diffusion assay *: Assay performed using agar diffusion method, Nt: Not tested

This is in accordance with literature reports; in fact phenolic compounds are the main chemical group responsible for the antimicrobial activity of plants including Rubiaceae and it well known that acetone or alcohol are the solvents of choice for the extraction of such components. Referring to Table 3 which displays some results recorded with Rubiaceae, available data do not show clearly whether the gram positive bacteria or the gram negative ones are more susceptible to the extract; but in general, the gram positive ones are found to be most susceptible (Karou et al., 2006). This selective susceptibility may be due to the biochemical composition of the cell wall. The gram positive bacteria have only an outer peptidoglycan layer which is not an effective barrier (Scherrer and Gerhardt, 1971). The Gram-negative bacteria have an outer phospholipidic membrane that make the cell wall impermeable to lipophilic solutes, while the porines constitute a selective barrier to hydrophilic solutes with an exclusion limit of about 600 Da (Nikaido and Vaara, 1985).

Cytotoxic activity: Medicinal plants are often assumed to be efficient and safe; however, there are some reports on poisonings consecutive to plant based-medicine administration (Fennell et al., 2004). Thus renewed interest is accorded to toxic effects of plant extracts. Many assays have been used to evaluate the toxicity plants. The main assays are based on the reduction of cell amount in cell culture and the results are expressed as IC₅₀ the drug concentration killing 50% of the cells. In a screening of some Nigerian antimalarial plants for in vitro cytotoxicity using brine shrimp IC₅₀ of 2.6, 383.9 and 9368 μg mL^-1 were recorded for Morinda lucida bark, Morinda lucida leaves and Nauclea latifolia bark extracts respectively, versus 449.1 μg mL^-1 for chloroquine phosphate. Indeed M. lucida was found to be less toxic than chloroquine phosphate and N. latifolia (Ajaiyeoba et al., 2006). The methanol extract obtained from Feretia apodanthera leaves, fractionated by silica gel chromatography and tested on TPH1 cells exhibited lower cytotoxicity with an IC₅₀ between 20 and 40 times higher than the IC₅₀ obtained on P. falciparum. The results of the effect on cell cycle and protein synthesis showed a decrease of cells in S phase and an accumulation in G2M phase, probably due to an inhibition of total protein synthesis Ancolio et al. (2002). Moreover using Allium cepa test, (Akintonwa et al., 2009) demonstrated that Morinda lucida at higher concentrations exhibited mitostatic effect and this may be due to the effect of the plant on the mitotic cell division process. However, the results of modified Ames test showed alteration of at least three biochemical characteristics of the normal organism, thus demonstrating mutagenicity.

Scientists now often associate the toxicity tests when looking for a particular biological activity; although some reports are systematically focussed on toxicology studies. The antimalarial activity is the main activity that has been associated with the cytotoxic activity. The activities are conducted on human cell or mammalian cell lines. Great IC₅₀ values have been recorded with crude extracts. Benoit-Vical et al. (1998) found 400 μg mL^-1 with aqueous extract of N. latifolia on human melanoma cells. According to Table 2, all IC₅₀ recorded were up to 40 μg mL^-1 except for the dichloromethane extract of Gardenia sokotensis which was found to be very toxic on W1-38 human fibroblasts (Jansen et al., 2010). The selective index is calculated by the ratio of the IC₅₀ and the IC₅₀ of the antimalarial test. This suggests the opportunity to continue the study with the fractionation of the extract.

Rubiaceae are continuously screened for their safety to ensure rational use in folk medicine. Some studies used animal models to better understand the in vivo manifestations of the toxic effects of the plants. Globally all results demonstrated the safety of tested Rubiaceae species supporting the continuous use of these plants in folk medicine. The in vivo toxicity of Mitracarpus scaber, Mitragyna inermis, Morinda lucida and Crossopteryx febrifuga are well documented. Looking for the possible hepatoprotective effect of Mitracarpus scaber decoction on carbon tetrachloride-induced acute liver damage in the rat Germano et al. (1999) found that treatment with the extract resulted in significant hepatoprotection against carbon tetrachloride-induced liver injury both in vivo and in vitro. In vivo, Mitracarpus scaber pretreatment reduced levels of serum Glutamate-Oxalate-Transaminase (GOT) and serum Glutamate-Pyruvate-Transaminase (GPT) previously increased by administration of carbon tetrachloride. In vitro the addition to the culture medium of Mitracarpus scaber extracts significantly reduced glutamate-oxalate-transaminase and lactate dehydrogenase activity resulting in a good survival rate for the carbon tetrachloride-intoxicated hepatocytes. A similar study previously conducted with Mitragyna inermis alkaloids extract resulted in the isolation of speciophylline as the main alkaloids of the leaves of the plant. The biological investigation showed that both speciophylline and total alkaloids extract were found to enhance bilary flow in female Wistar rats. In addition total and conjugated bilirubin were increased significantly, while GOT, GPT, alkaline phosphatase and total cholesterol decreased indicating an obvious hepatic cellular activity induced by the alkaloids without cellular necrosis. The authors concluded that alkaloid extract and particularly speciophylline may act as choleretic drugs (Toure et al., 1996).

Acute and chronic toxicity of the hydroethanolic extract of Mitragyna inermis leaves were performed in rats, according to the recommendations the French Drug Office. No animal died and no behavioral signs of acute toxicity were observed after two dosages (300 mg kg^-1 and 3 g kg^-1) were administered to animals. In addition, no changes in body weight and no macroscopic abnormality in examined organs after 28 days chronic toxicity follow up (Monjanel-Mouterde et al., 2006). However, Konkon et al. (2008) demonstrated that the administration of 300, 2000 and 5000 mg kg^-1 aqueous extract of leaves of the plant was lethal for the inoculated animals. Indeed the aqueous leaf extract of M. inermis should be used with some degree of safety by oral route. The maximal dose seems to be 300 mg kg^-1. However, the methanolic extract of C. febrifuga seemed to be less toxic, since the extract did not produce severe toxicity at dose lower than 500 mg kg^-1 body weight (Salawu et al., 2009). Recently, the aqueous extract of N. pobeguinii was found to be non toxic in mice model. Thus levels of creatinin, urea, GOT and GPT remained unchanged after treatment; in addition, no acute toxicity was observed in mice and no significant macroscopic or microscopic lesions were observed in organs neither after a single 2 g kg^-1 oral dose, nor after 4 weekly doses (Mesia et al., 2010).

Antioxidant activity and anti-inflammatory activity: Antioxidant and radical scavenging properties of plants are subject to intensive research. Indeed Rubiaceae are continuously screened for these pharmacological properties. Maiga et al. (2006) found that the methanol extract of the seed of C. febrifuga had a moderate free radical scavenging using the stable free radical diphenylpicrylhydrazyl. However, the lipophilic fraction that was found to have no scavenging activity highly inhibited the soybean 15-lipoxygenase. In a similar study, Dongmo et al. (2003) demonstrated that the methanolic extract of Mitragyna cilita had not inhibitory effect on the 5-lipoxygenase, although the extract reduced carrageenin-induced paw oedema in rat showing an effective anti-inflammatory effect. The author suggested that the active compounds may exert the activity on another sites implicated in the anti-inflammatory process. This was confirmed by the observed analgesic activity of the same methanol extract through significant increase of the threshold of sensitivity to pain in the rats with salicylates as standard analgesic. Similar results were recorded with methanolic extract of C. febrifuga; indeed the extract significantly diminished acetic acid-induced writhes in mice and increased the pain threshold in rats dose-dependently. It also demonstrated significant antipyretic and anti-inflammatory activities in mice and rats in a dose-related manner (Salawu et al., 2008). Aqueous extract of the root bark of N. latifolia was evaluated for its anti-nociceptive, anti-inflammatory and anti-pyretic activities in mice and rats. The results showed that the extract significantly attenuated writhing episodes induced by acetic acid and increased the threshold for pain perception in the hot-plate test in mice, dose-dependently. The product also remarkably decreased both the acute and delayed phases of formalin-induced pain in rats and also caused a significant reduction in both yeast-induced pyrexia and egg-albumin-induced oedema in rats. These effects were produced in a dose-dependent manner (Abbah et al., 2010).

Antidiabetic activity: Following the traditional usage, some Rubiaceae species including Morinda lucida and Nauclea latifolia have been screened for antidiabetic activity. Gidado et al. (2008) screened N. latifolia for it fasting blood glucose lowering effect in normoglycaemic and streptozotocin-diabetic Wistar albino rats. The aqueous and ethanolic extracts significantly lowered the fasting blood glucose levels of the diabetic rats in a dose-dependent manner; however, the aqueous extract did not significantly lower the glucose levels of normoglycaemic rats. The hypoglycaemic and antihyperglycaemic potentials of the aqueous and ethanolic extracts were comparable to that of glibenclamide supporting the traditional use of the plant in the treatment of diabetes mellitus. Olajide et al. (1999) previously found that the methanol extract of the Morinda lucida exerted a dose-dependent hypoglycaemic activity in normal rats within 4 h after oral administration. In hyperglycaemic rats, the extract produced a significant anti-diabetic effect from day 3 after oral administration. Furthermore, the aqueous extract of the roots of the plant, exhibited potent hypoglycaemic effects in both normoglycemic and alloxan-induced diabetic mice by oral administration. This effect was dose-dependent and more potent than that observed with chlorpropamide (l-(p-chlorobenzene-sulphanyl)-3-propylurea) (Kamanyi et al., 1994).

Other biological activities: Rubiaceae species have been screened for many other pharmacological properties including antispomodic, antihyperthermic, anticonvulsive and antipyretic activities. The in vitro antispasmodic activity of Morinda morindoides leaves extracts was evaluated on acetylcholine and the depolarized KCl solution induced contractions on guinea-pig isolated ileum. The issue of the study revealed that M. morindoides leaves possess spasmogenic and spasmolytic properties that can at least explain and support its traditional use against constipation and diarrhoea, respectively (Cimanga et al., 2010). This property was already described for C. febrifuga and N. latifolia. Polyphenol extracts of these plants exhibited more than 70% inhibition of contractions on isolated guinea-pig ileum; in addition to inhibit Entamoeba histolytica growth (Tona et al., 2000).

Extract Morinda lucida was screened with 12 other Congolese medicinal plants for their antidrepanocytary activity through the ability of the extracts to normalize the SS blood erythrocytes. The results showed normalization rate (45%) of the methanol extract of leaves and bark, however, the aqueous extract failed in normalizing the cells (Mpiana et al., 2007). The leaf extract of the plant investigated for possible antispermatogenic activity did not cause any changes in body and somatic organ weights, but significantly increased the testis weight. The sperm motility and viability and the epididymal sperm counts of rats treated for 13 weeks were significantly reduced. Sperm morphological abnormalities and serum testosterone levels were significantly increased. There were various degrees of damage to the seminiferous tubules. The extract also reduced the fertility of the treated rats by reducing the litter size. Reversal of these changes, however, occurred after a period of time (Raji et al., 2005). Similar effects were observed with the aqueous extract of Fadogia agrestis. In addition, the extract induced significant increases in the prostrate/body weight ratio, citric acid concentration and acid phosphatase activity at all the dose regimen and only at 50 and 100 mg kg^-1 body weight dose regimen for calcium and phosphate, while pH was not altered. There was no recovery on prostatic parameters except the citric acid content at 18 mg kg^-1 body weight (Yakubu et al., 2007).

The decoction from the bark of N. latifolia tested for its anticonvulsant, anxiolytic and sedative activity in mice was found to increase the total sleep time induced by diazepam in mice model and to protect mice against maximal electroshock-, pentylenetetrazol- and strychnine-induced seizures. In addition, turning behaviour induced by N-methyl-D-aspartate was inhibited. The extract antagonized, in a dose-dependent manner, stress-induced hyperthermia and reduced body temperature (Ngo Bum et al., 2009). The anthelmintic efficacy of aqueous extract of stem bark of the plant was investigated in sheep with natural acute and sub-acute parasitic gastro-enteritis due primarily to mixed nematode species. Graded doses of the extract improved haemoglobin and leucocytosis values in worm-infected sheep and significantly reduced faecal egg counts in infected animals. The percentage reduction by 1600 mg kg^-1 of the extract was comparable to that of 5 mg kg^-1 of albendazole (Ademola et al., 2007; Onyeyili et al. (2001). Njamen et al., (2008) evaluated the in vitro estrogenic activity of the methanol extract of the plant using the yeast test-system. The extract yielded interesting activity and was then further investigated on alkaline Phosphatase induction in Ishikawa cells. The results showed significant stimulatory effects at 10 and 100 mg mL^-1 doses. In vivo the extract had not effect on the uterine epithelial height on ovariectomised rats, although the administration of 200 mg kg^-1 increased vaginal epithelial height by 15.64%, confirming the estrogenic activity of the plant.

The evaluation of the neuropharmacological effects of the aqueous extract of N. latifolia root bark in rodents were assayed by measuring the effects on the Spontaneous Motor Activity (SMA), exploratory behaviour, pentobarbital sleeping time, apomorphine-induced stereotypic behaviour and motor coordination (rota-rod performance). The extract significantly decreased the SMA and exploratory behaviour in mice and prolonged pentobarbital sleeping time in rats dose-dependently. The intensity of apomorphine-induced stereotypy was also attenuated dose-dependently in mice, but no effect on motor coordination as determined by the performance on rota-rod was recorded indicating the presence of psychoactive substances in the aqueous extract of the root bark of N. latifolia (Amos et al., 2005).

Looking for possible hypotensive, cardiotropic and vasodilatory properties M. inermis, Ouedraogo et al. (2004) found that the aqueous extract of the plant produced a concentration-dependent ex vivo increase in cardiac contractile response and coronary flow but did not modify heart rate in the rat. Adverse effects were observed with the extract of N. latifolia which was found to reduce systolic, diastolic and mean arterial pressure in normotensive and in one kidney one clip hypertensive rats in a dose dependant manner. The extract also reduced the heart rate of normotensive and hypertensive rats. The reduction in blood pressure and heart rate was not affected by prior treatment with atropine or promethazine (Nworgu et al., 2008). In the case of M. inermis, the extract produced relaxation in isolated porcine coronary artery at concentration up to 3 mg mL^-1 that was exclusively dependent on the presence of endothelium. This relaxation involved partial depolarization and NO synthase inhibitor-sensitive mechanisms but was not sensitive to the blockade of cyclo-oxygenase pathway. However, the relaxant effect was not dependent on the presence of endothelium in rat tail artery.

CONCLUSION

The present review discussed the significance of Rubiaceae as a valuable source of new leads for medical purposes. Reports for biological activity of Rubiaceae species are numerous, but phytochemical investigations have been conducted only on a few species such as N. latifolia, N. pobeguinii, M. inermis, P. bussei and P. longiflora. Indole alkaloids seem to be typical of the family as they were detected in several species. Correlation between the traditional uses and the pharmacological activities has been observed and described in the present review. Significant activity of the alkaloids isolated from certain species against Plasmodium has been reported. Crude extracts of these plants have been found to have antibacterial, antidiabetic, anti-inflammatory; antioxidant activities and the lack of toxicity have been reported in some cases. However, referring to current situation, HIV is the main major public health problem and much effort is made by scientists in this topic. HIV control efforts may include the attempt to seek for effective agents able to kill the virus itself, agents able to boost up the immune system and agents able to treat opportunistic infections. According to online published data Rubiaceae exhibited antimicrobial activity against several pathogens including AIDS opportunistic ones. Indeed, antimicrobial property of Rubiaceae may be useful tool in treating opportunistic infection. Therefore, the new challenge is the investigation for immunomodulatory and antiviral activities of Rubiaceae, considering the lack of published data in this matter.