The Golden Roots of Cryptolepis sanguinolenta - ACS Publications

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The Golden Roots of Cryptolepis sanguinolenta Alfred Ampomah Appiah Centre for Scientific Research into Plant Medicine P. O. Box 73, Mampong-Akuapem, E/R, Ghana

The root of the plant Cryptolepis sanguinolenta (Lindl.) Schlt (Periplocaceae) is used in traditional African medicine to treat a variety of diseases including malaria, jaundice, hepatitis, urinary tract infections, hypertension, inflammatory conditions and stomach ache. Extracts of the roots are also used as a tonic often taken daily for years without evidence of toxicity. Various studies indicate that the crude extracts as well as the isolated alkaloidal constituents of the plant possess a number of interesting pharmacological properties. The focus of this overview is to highlight the potential of Cryptolepis sanguinolenta in modern health care.

© 2009 American Chemical Society

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In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Description C. sanguinolenta (Lindl.) Schlt (Periplocaceae) is a shrub that grows in the rainforest and the deciduous belt forest found in West Africa. Related species appear in the east and southern regions of the continent (1). The plant is a slender climber up to 8 meters high; with blood-red exudates; leaves elliptic, oblong-elliptic, or ovate, acutely and shortly acuminate, rounded 2.5 – 7 cm long and 1 - 3 cm broad; flowers greenish-yellow (2). The fruits are paired in linear follicles and are horn-like. The seeds are oblong in shape, small (averaging, 7.4 mm in length and 1.8 mm in the middle), and pinkish, embedded in long silky hairs. Dried C. sanguinolenta roots have sweet fragrance and a bitter taste. The surface of the cut dried roots show a bright yellow color (3). Ethnomedical Use C. sanguinolenta is one of the important herbs in West African traditional medicine. According to Silva et al the root of this plant has been used in Guinea Bissau by Fulani traditional healers for the treatment of jaundice and hepatitis. (4). Kerharo et al (as cited in Addy 2003) have also reported that the infusions of the roots are used in the treatment of stomach and intestinal disorders in Zaire and the Casamance district of Senegal (3). In Ghana, the root is a popular antimalarial. The roots have also been used to treat urinary and upper respiratory tract infections (5). Unconfirmed claims by some traditional medical practitioners in Ghana suggest that C. sanguinolenta root extract enhances sexual performance (6) and it is also effective in the management of diabetic symptoms (7), hemorrhoids (8), breast tumors and related disease (9). An alcoholic extract of the root is also used in Ghana as a tonic to strengthen metabolism (7). Tona et al have reported that an extract of C. sanguinolenta is used in Congolese traditional medicine for the treatment of diarrhea (10). According to Iwu et al., the plant is mainly used for the treatment of fevers; it is also used to treat urinary tract infections, especially Candida (1). Iwu et al. has also reported that the plant has other uses which include the treatment of inflammatory conditions, hypertension, microbial infections and stomach ache (1). Chemistry Most of the compounds isolated from C. sanguinolenta are alkaloids and are analogs of indolo[3,2-b]quinoline (11). The major alkaloidal component of the plant is cryptolepine (12). In addition to cryptolepine, comparatively small quantities of a number of related alkaloids have been isolated from the plant. These include 11-hydroxycryptolepine, cryptoheptine, isocryptolepine, quindoline, biscryptolepine, cryptoquindoline, cryptolepicarboline, cryptospirolepine. Quindolinone, cryptotakienine, and cryptomisrine (11,13-16) (Figure 1).


233

OH

N

N

N

N

N CH3

CH 3

Cryptolepine

11-Hydroxycryptolepine

Isocryptolepine (Cryptosanguinolentine)

O


CH3

N

H N

N

H N

N H 3C

N H

N

Quindoline

OH

Quindolinone

Cryptoheptine

CH 3 CH3

N

N

N

N

N

N

N

N

N N

CH 3

CH 3

Cryptotackieine

Biscryptolepine

Cryptoquindoline

CH3

N

N

N H

N

N

N

O

N

N N CH 3

Cryptolepicarboline

O

H

H N

N H 3C

Cryptospirolepine

N

Cryptomisrine

Figure 1. Structures of compounds (alkaloids) isolated from C. sanguinolenta.

Pharmacological Properties Extracts of C. sanguinolenta roots and/or cryptolepine have been found to possess several interesting biological/pharmacological properties. These include: anti-plasmodial/anti-malarial (17), antimicrobial (18-23), vasodilatation, vasoconstriction, anti-hypertensive, noradrenoceptor antagonism (24), antimuscarinic (25), anti-hyperglycemic (26), anti-inflammatory (27), and anti-viral (28) activities.



234 Cimanga et al (17) assessed the anti-plasmodial (anti-malarial) activity of three different extracts and four alkaloids from the root back of C. sanguinolenta in vitro against P. falciparum D-6 (chloroquine-sensitive strain), K-1, and W-2 (chloroquine-resistant strains). They observed that cryptolepine, cryptolepine hydrochloride, 11hydroxycryptolepine, and neocryptolepine showed a strong antiplasmodial activity against P. falciparum chloroquine-resistant strains. Quindoline was less active. The highest activity was obtained with cryptolepine. An in vivo tests on infected mice showed that crytpolepine, when tested as its hydrochloride, exhibited a significant chemosuppressive effect against Plasmodium berghei yoelii and Plasmodium berghei berghei, while Cryptolepine had the same effect against P. berghei yoelii only. 11-hydroxycryptolepine and Quindoline did not show activity in the in vivo test system. In general, they could not deduce any correlation between the in vitro and the in vivo antiplasmodial activity of the test samples. They were of the view that, although cryptolepine or its hydrochloride is less active than known antimalarials such as mefloquine or artemisinin in vitro, and less active than chloroquine in vivo, their result provide some rational evidence for the use of C. sanguinolenta for the treatment of malaria in traditional medicine in some African countries; and that cryptolepine or related alkaloids may provide useful lead structures for the development of a new class of synthetic antimalarials. Tackie et al have patented some antimalarial compounds isolated from C. sanguinolenta (29). As a response to increasing levels of antimalarial resistance, The World Health Organization (WHO) recommends that treatment policies in all countries experiencing resistance of Plasmodium falciparum to conventional monotherapies should be combination therapies, preferably those containing artemisinin derivatives. Currently WHO recommends the following therapeutic options: artesunate-sulfadoxine/pyrimethamine, artesunate-amodiaquine, artesunate-mefloquine, sulfadoxine/pyrimethamine-amodiaquine and artemether-lumefantrine (30). The root extracts/isolates of C. sanguinolenta may be used in combination therapy of malaria. Other in vitro studies have shown that extracts of C. sanguinolenta have anti-microbial activity. Activity against Staphylococcus aureus, E. coli, Candida albicans and enteric pathogens have been reported (4,18-22). Paulo et al (22) determined the Minimum Inhibitory Concentrations (MICs) of the ethanol and the aqueous extracts of C. sanguinolenta roots, and that of cryptolepine for 65 strains of Campylobacter jejuni, 41 strains of Campylobacter coli isolated from sporadic cases of gastroenteritis in Portugal and 86 strains of Vibrio cholerae isolated from patients with enteric infections in Angola, Brazil and Portugal. They found that the ethanol extract activity against Campylobacter strains (MIC = 25 μg/mL) is higher than that of co-trimoxazole and sulfamethoxazole and Campylobacter strains susceptibility for cryptolepine (MIC = 12.5 μg/mL) is about the same as that of ampicillin. The ethanol extract and cryptolepine show some activity against the Vibrio cholerae strains, although their activities are lower than that of tetracycline. They concluded that, the roots of C. sanguinolenta could be a therapeutic alternative for bacterial etiologic diarrhea.



235 Gibbons et al (23) assessed the activity of cryptolepine hydrochloride against the fast growing mycobacterial species Mycobacterium fortuitum, which has been shown to be of use in the evaluation of antitubercular drugs. They observed that the minimum inhibitory concentration (MIC), 16 µg/mL, of the compound was low. This prompted further evaluation against other fast growing mycobacteria namely, M. phlei, M. aurum, M. smegmatis, M. bovis BCG and M. abcessus and the MICs ranged over 2-32 µg/mL for these species. In conclusion, they stated that, the strong antimycobacterial activity of the compound and the need for new antibiotics with activity against Mycobacterium tuberculosis, coupled with the ethnobotanical use of C. sanguinolenta extracts to treat infections, highlight the potential of the cryptolepine template for development of antimycobacterial agents. Bamgbose and Noamesi (as cited in Tackie et al 1993) reported that cryptolepine possess hypotensive and vasoconstritive activity. In addition, these same authors observed that cryptolepine hydrochloride is a noradrenoceptor antagonist in the isolated rat vas deferens. Raymond-Hamet (as cited in Tackie et al 1993) reported that administration of cryptolepine hydrochloride to dogs at a dosage of 15-30 mg/kg produced marked hypothermia and decreased the hypertensive and renal vasoconstrictive actions of epinephrine; and that iv administration of 5 mg/kg of the alkaloid produced a marked and protracted hypotensive response in the vagotomised dog with a corresponding decrease in renal volume. They inferred that the pronounced vasodilatation produced by the alkaloid was the cause of these effects (24). Cryptolepine seems to have a yohimbine-like activity: a selective α2-receptor antagonist. By blocking α2receptors, while sparing α1-receptors yohimbine increases noradrenaline release, and produce sympathomimetic effects in some organs. Blockade of postsynaptic α2-receptors which occurs in blood vessels and some other organs causes a block of sympathetic responses; so the overall effects are complex. Vasodilatation and a fall in blood pressure usually predominate, and its vasodilator effect has given yohimbine fame as an aphrodisiac (31). Cryptolepine (3-30 µM) and the alkaloid fraction of C. sanguinolenta (310 μg/mL) antagonized muscarinic effects at M1 receptors in rabbit vas deferens, M2 receptors in guinea-pig atria, and M3 receptors in guinea-pig ileum. The experiments, using N-methylatropine as reference drug, showed a significant antimuscarinic activity for both cryptolepine and the alkaloid fraction, but no appreciable receptor subtype selectivity. Cryptolepine was determined as the antimuscarinic principle of C. sanguinolenta (25). The antimuscarinic properties of Cryptolepine suggests that C. sanguinolenta may be useful in one or more of the following disease conditions: urinary incontinence, asthma, peptic ulcer, parkinson’s disease and motion sickness. The anti-hyperglycemic property of cryptolepine has been shown as enhanced insulin-mediated glucose disposal in a mouse model of diabetes and in an in vitro system using the 3T3-L1 glucose transport assay, indicating an effect on Type 2 diabetes (26). Some hypoglycemic agents isolated from C. sanguinolenta have been patented by Luo et al (32). Cryptolepine has been found to possess anti-inflammatory effects in the carrageenan-induced paw edema in rats (27); and Iwu (as cited in Iwu et al 1999) has also reported that the compound has shown histamine antagonism (1).


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Toxicity In one of their studies, Ansah et al (9) examined the in vitro toxicity of the aqueous extract of C. sanguinolenta (CSE) and the alkaloid cryptolepine (CLP) using V79 cells, a Chinese hamster lung fibroblast, and a number of organspecific human cancer cell lines. CSE and CLP caused a dose- and timedependent reduction in viability of the V79 cell line. In a V79 cell mutation assay (hprt gene), CSE (5–50 µg/mL) only induced mutation at the highest dose employed (mutation frequency 4 and 38 mutant clones per 106cells for control and CSE, respectively), but CLP (0.5–5.0 µM) was not mutagenic. They observed that the mutagenic dose of CSE was very toxic (less than 15% cell survival) and concluded that the poor genotoxicity of CSE and CLP coupled with their potent cytotoxic action support their anticancer potential. They also indicated that the remarkable similarity in the cytotoxic profiles of the two agents would suggest that CLP is responsible for the activity of CSE, consistent with a previous report that CLP is the major alkaloid of the root extract. In a study to establish the molecular basis for the diverse biological effects of cryptolepine hydrochloride, Bonjean et al (33) observed that DNA is the primary target of cryptolepine. They indicated that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. Their study also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. Using the fluorescent probe 2', 7'-dichlorofluorescein-diacetate (DCFHDA) to measure intracellular changes in reactive oxygen species (ROS) Ansah et al (34) observed that both the aqueous anti-malarial formulation of C. sanguinolenta (CSE) and cryptolepine (CLP) caused a dose-dependent increase in ROS production, which reduced significantly following pre-treatment with Nacetylcysteine, an anti-oxidant. They suggested that reactive oxygen species generation is associated with cryptolepine cytotoxicity. In another study, Cimanga et al (35) isolated six of the Cryptolepis alkaloids, identified them spectroscopically as cryptoquindolinine, quindoline, neocryptolepine, cryptolepine, 11-hydroxycryptolepine and biscryptolepine, and tested their interaction with the xanthine–xanthine oxidase enzyme system. 11hydroxycryptolepine was shown to inhibit xanthine oxidase and act as a scavenger of superoxide anions. The other alkaloids were devoid of effect in both assays at the highest test concentration of 100 mM. They concluded that these findings suggest the importance, in the manifestation of both activities, of the hydroxyl group present in 11-hydroxycryptolepine but not in the other alkaloids. This result is interesting because if the major metabolite of C. sanguinolenta alkaloids after oral administration happens to be 11hydroxycryptolepine, it is possible that the extract of the plant may have a net effect as a scavenger of superoxide anions. Its use as a daily tonic may therefore be justified.


237


Assay It can be inferred from Appiah’s work (36) that the Cryptolepine Base Equivalent (i.e. cryptolepine equivalent of the total alkaloidal content) of C. sanguinolenta roots is approximately 0.6%w/w. This means that a dose of Phyto-laria (3) which is a tea-bag containing 2.5g of powdered C. sanguinolenta roots is equivalent to 15mg of the total alkaloidal extract. Since this product is recommended to be taken three times daily, it implies that the total daily dose is equivalent to 45mg of C. sanguinolenta alkaloids. This works out to a daily dose of 0.6mg C. sanguinolenta alkaloids/kg body weight (taking the average body weight to be 70 kg). In another study, Appiah and Sittie (37) found that the ratio of the total alkaloid in a daily dose of four different products of C. sanguinolenta roots on the Ghanaian market is 3:4:4:10. The daily maximum dose of C. sanguinolenta alkaloids (in a product of C. sanguinolenta root) recommended by herbal practitioners in Ghana is far less than 2.0 mg/kg body weight, and this is normally administered orally in three divided doses. The clinical doses are therefore very low as compared to the concentrations used in various in vitro/in vivo experiments including that of the LD50 determination which was found to be 146 ± 49.3 mg of cryptolepine/kg in the male albino mouse after ip injection (38). This is probably one of the reasons why no major adverse effect has been observed over the long period of use of C. sanguinolenta. It is noteworthy that Luo et al. (as cited in Addy 2003) report the use of C. sanguinolenta extract as a tonic, often taken daily for years without evidence of side effects or toxicity (3). For the past 15 years, Gyapong (39), presently about 58 years old, has been taking C. sanguinolenta root decoction almost everyday; and he looks younger and healthier than most of his peers. Unwanted/Adverse effects The unwanted/adverse effects that might occur following an overdose of C. sanguinolenta extract administered orally include blurred vision, dry mouth, constipation, stomach upset, dizziness, postural hypotension and confusion. These effects are generally reversible and are probably due to the vasodilatory and the antimuscarinic effects of the plant extract.

Conclusion The pharmacological properties of cryptolepine confirm most of the ethnomedical uses of C. sanguinolenta by West African natives. Administration of the aqueous extract of C. sanguinolenta seems to exert significant and desirable physiological and clinical effects. The plant has a great potential for the development of essential medicines including anti-cancer agents. For instance, taking into account the unwanted side effects of some antihypertensive and anti-diabetic agents which include gout, erectile dysfunction and asthma, C. sanguinolenta could be developed as an anti-hypertensive and/or


238 anti-diabetic with a possible desirable side effect as an enhancer of sexual performance.

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The Golden Roots of Cryptolepis sanguinolenta - ACS Publications

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