Antiplasmodial Studies Within the Plant Family Amaryllidaceae

Abstract

Plants have long served as a first line of defense response to malaria. They have also spawned several classes of antimalarial drugs such as quinine and artemisinin. However, most if not all of these drugs have succumbed to multidrug resistance, thus reigniting interest in the identification of novel chemotherapies against this parasitic disease. The starting point for many of such endeavors lies with the plants themselves whose extracts have served as herbal remedies, which originate from traditional medicine (TM). Several species of the Amaryllidaceae have been shown to have such functions in TM. This survey examines those plants of the family, which have hitherto been examined for antiplasmodial effects against the malarial parasite Plasmodium falciparum. Also considered are the alkaloid constituents of these plants, which have demonstrated activities against various strains of the pathogen. Particular emphasis is made on those plants which both demonstrate such activity as well as have a place in traditional therapies for malaria.

There is fossil evidence to support the existence of malaria as far back as the Palaeogene period of the geological time scale some 60 million years ago. ¹ Ancient texts also verify the prevalence of malaria in Mesopotamia over 3000 years ago, in Egypt around 3000 years ago, in China during the Han dynasty (~2500 BCE), and in India through Vedic script during the period 1500 to 800 BCE. ¹ Malaria is thought to have originated in Africa and followed human expansion to every continent of the globe apart from Antarctica. ¹ This tropical disease is caused by a number of organisms of the Plasmodium group of protozoan parasites, mainly Plasmodium falciparum. ² Mosquitoes belonging to the genus Anopheles such as A. gambiae are the chief vectors of transmittance for the disease. ³ The pathogen requires both a human target and insect vector to survive since the different phases of its life cycle may be carried out in either of these hosts. ³ Following intensive preventive measures involving the use of insecticide sprays, insect repellents, mosquito nets, and prophylactic drugs, malaria has been successfully controlled in most parts of the world. ² Although it is still prevalent in some parts of the Americas and Asia, its highest incidence is in Africa along a broad band permeating either side of the equator. ⁴ Here it is responsible for over 90% of the more than 200 million cases diagnosed annually. ⁴

Plants have had a long and rich cultural association with traditional approaches to malaria wherein over a thousand individual taxa globally are identifiable with such systems of remediation. ⁵ They have also afforded a rich and diverse source of antiplasmodial substances, incorporating most classes of natural products, that is second to none. ⁶ Most notable among these are the Cinchona alkaloid quinine and the Artemisia sesquiterpene lactone artemisinin. ⁷ The discovery of both of these compounds is based on centuries-old indigenous knowledge systems entrenched in Quechua and Chinese cultures, respectively, which have since been incorporated into contemporary medicine. ⁷ Multidrug resistance and the diminishing efficacies of existing drugs are the key factors, which have served to rekindle interest in plants as a source of new antimalarial targets. ⁷ Furthermore, it is estimated that a relatively small percentage (<20%) of the floral biodiversity worldwide has been examined for potential therapeutic effects. ⁵ The plant family Amaryllidaceae has recently been shown to be a novel source of antiplasmodial constituents evolving out of its crinane, lycorane, and minor alkaloid groups. ^{8 -10} This mini-review details those members of the Amaryllidaceae whose extracts have been examined for antiplasmodial activities against various strains of the malaria parasite Plasmodium falciparum. Particular attention is paid to those species, which have documented uses in traditional medicine (TM) for the treatment of this disease. Attempts are also made to explain the activities of the extracts via the phytochemical principles they have yielded. These were seen to belong to the isoquinoline alkaloid constituents, which characterize the plant family Amaryllidaceae.

The warm, humid, and lush conditions typifying equatorial Africa afford the ideal breeding grounds for the malarial parasite. ⁴ Providing the necessary care to stricken patients is often impeded by ailing healthcare infrastructure, limited access to medical facilities, ill-equipped medical personnel, as well as a shortage of essential medicines. ⁴ Nonetheless, there is a rich culture of TM within this malaria zone, with up to 80% of the population in some countries reliant on this form of medicine for their primary healthcare needs. ¹¹ Furthermore, the region is home to the Paleotropical floral kingdom, which is recognized for its exceptionally high diversity and endemism. ¹² In particular, significant successes have been realized in the use of these plants and their products in traditional remedies for malaria. ¹² There were 8 Amaryllidaceae plants identified in the literature whose extracts and preparations have been exploited in ethnic medicine for the treatment of malaria. Of these, 3 (Boophone disticha (L.f.) Herb., Crinum bulbispermum (Burm.f.) Milne-Redh. & Schweick., and C. macowanii Baker) were from South Africa, 2 (C. amabile Donn ex Ker Gawl. and Pamianthe peruviana Stapf) were from Sudan, while the remaining 3 (Crinum asiaticum L., C. erubescens L.f. ex Aiton, and C. zeylanicum (L.) L.) had their origins in India, Haiti, and the Dominican Republic, respectively. ^{13 -18} A further 3 plants including C. firmifolium Baker, Hippeastrum parodii Hunz. & A.A.Cocucci, and Zephyranthes carinata Herb. from Madagascar and Argentina, respectively, have been indicated in the remediation of parasitic infections which could also include malaria. ^19,20

Interestingly, the first antimicrobial screen recorded on the Amaryllidaceae was in relation to its antiplasmodial effects. ²¹ In this seminal work, 44 different plants from 18 genera were examined for activity against the avian malarial parasite Plasmodium gallinaceum. ²¹ As such, malarial infections were produced in healthy 7-day-old chicks via inoculation with a quantity of sporozoite suspension equivalent to roughly that which would be obtainable from a single mosquito. ²¹ Ethanol extracts of the plants were then administered via the subcutaneous injection route immediately after sporozoite inoculation and continued for a period of 3 days thereafter. ²¹ In the event good activities were observed for 10 plants from 8 genera of the Amaryllidaceae. ²¹ The best overall activity was observed for the native South American species Zephyranthes drummondii D.Don wherein a minimum effective dose (MED) of 3.2 mg/kg was determined using quinine as the reference standard (Table 1). ²¹ Other activities of note included those for Hippeastrum puniceum (Lam.) Voss (MED 25 mg/kg), Hippeastrum vittatum (L'Hér.) Herb. (MED 80 mg/kg), Hymenocallis americana (Mill.) M.Roem. (MED 50 mg/kg), and Hymenocallis coronaria (Leconte) Kunth (MED 100 mg/kg). ²¹ Although the activities for Boophone disticha and C. erubescens were in this instance not noteworthy, ²¹ both of these plants as alluded to above have reputed uses in the traditional remediation of malaria. ^{13 -15}

Table 1

Antiplasmodial Activities of Extracts Derived From Various Species of the Amaryllidaceae.

No.	Plant	Extract	Pathogen/strain	Activity (IC50, μg/mL) a	Ref.
1	Amaryllis belladonna	MeOH	Plasmodium falciparum Dd2	Active b	27
2	Boophone disticha c	EtOH	Plasmodium gallinaceum	Not active	21
3	Brunsvigia littoralis	EtOH EtOH	Plasmodium falciparum D10 Plasmodium falciparum FAC8	Active b Active b	26 26
4	Crinum amabile c	EtOH EtOH MeOH MeOH	Plasmodium falciparum D-6 Plasmodium falciparum W-2 Plasmodium falciparum K1 Plasmodium falciparum NF54	ED50 1.6 µg/mL ED50 4.2 µg/mL Not active Not active	22 22 16 16
5	Crinum asiaticum c	CHCl3 CHCl3	Plasmodium falciparum RKL-2 Plasmodium falciparum 3D-7	5.78 µg/mL 3.18 µg/mL	25 25
6	Crinum bulbispermum c	DCM EtOAc	Plasmodium falciparum FCR-3 Plasmodium falciparum FCR-3	IC50 0.38 µg/mL IC50 0.08 µg/mL	23 23
7	Crinum erubescens c	EtOH MeOH	Plasmodium gallinaceum Plasmodium falciparum Dd2	Not active <1.25 µg/mL	21 24
8	Crinum firmifolium c	−	−	−	19
9	Crinum macowanii c	H2O DCM/MeOH	Plasmodium falciparum D10 Plasmodium falciparum D10	IC50 25 µg/mL IC50 26 µg/mL	17 17
10	Crinum zeylanicum c	−	−	−	15
11	Hippeastrum parodii c	−	−	−	20
12	Hippeastrum puniceum	EtOH	Plasmodium gallinaceum	MED 25 mg/kg	21
13	Hippeastrum vittatum	EtOH	Plasmodium gallinaceum	MED 80 mg/kg	21
14	Hymenocallis americana	EtOH	Plasmodium gallinaceum	MED 50 mg/kg	21
15	Hymenocallis coronaria	EtOH	Plasmodium gallinaceum	MED 100 mg/kg	21
16	Leucojum aestivum	EtOH EtOH	Plasmodium falciparum T9.96 Plasmodium falciparum K1	IC50 1.47 µg/mL IC50 1.75 µg/mL	29 29
17	Narcissus broussonetii	EtOAc EtOAc	Plasmodium falciparum K1 Plasmodium falciparum NF54	Not active Not active	28 28
18	Narcissus tazetta	EtOH EtOH	Plasmodium falciparum T9.96 Plasmodium falciparum K1	IC50 2.55 µg/mL IC50 3.31 µg/mL	29 29
19	Pamianthe peruviana c	MeOH MeOH	Plasmodium falciparum K1 Plasmodium falciparum NF54	IC50 0.63 µg/mL IC50 1.11 µg/mL	16 16
20	Pancratium maritimum	EtOH EtOH	Plasmodium falciparum T9.96 Plasmodium falciparum K1	IC50 1.56 µg/mL IC50 1.83 µg/mL	29 29
21	Zephyranthes carinata c	−	−	−	20
22	Zephyranthes drummondii	EtOH	Plasmodium gallinaceum	MED 3.2 mg/kg	21

ED₅₀, effective dose; MED, minimum effective dose; IC₅₀, half-maximal inhibitory concentration.

a

Activities described as IC₅₀s (μg/mL) unless otherwise stated.

b

Numerical data not provided.

c

Plants reputedly used in traditional medicine for malaria.

In spite of these promising early findings the antimalarial potential of the Amaryllidaceae remained dormant for nearly 5 decades when studies on their antiplasmodial activities were reinitiated in 1993. ²² In this study ethanolic bulb extracts of C. amabile specimens collected in Thailand were subjected to screening measures against the chloroquine-sensitive D-6 and chloroquine-resistant W-2 strains of Plasmodium falciparum, where the effective doses (ED₅₀s) were determined as 1.6 and 4.2 µg/mL, respectively. ²² The nearly 3-fold better activity observed for the W-2 strain over the D-6 strain is in this instance noteworthy. ²² In search of the bioactive constituents, it was shown that the alkaloids lycorine (1) and augustine (2) (Figure 1) exhibited good activities against both the D-6 (ED₅₀s 0.32 and 0.14 µg/mL, respectively) and W-2 (ED₅₀s 0.30 and 0.18 µg/mL, respectively) strains. ²² In perspective, however, these activities paled in comparison to those calculated for the artemisinin standard, which were 0.6 and 0.5 ng/mL against the D-6 and W-2 strains, respectively. ²² These results could be used to justify the usage of C. amabile bulb preparations in traditional remedies for malaria. ¹⁶

Figure 1

Antiplasmodial alkaloids from Amaryllidaceae plants.

A separate examination of C. amabile involved a bulb collection that was made in Africa. ¹⁶ This investigation examined the antiplasmodial activities of 19 plant species that are known to be used in Sudanese TM for malaria and other related ailments. ¹⁶ Of these, C. amabile and Pamianthe peruviana were members of the Amaryllidaceae with recorded uses for such parasitic diseases. ¹⁶ Among other assays, methanol extracts of the plants were screened against the malarial parasite Plasmodium falciparum strains K1 (multidrug-resistant) and NF54 (chloroquine-sensitive). ¹⁶ Of the 2 Amaryllidaceae species, only Pamianthe peruviana was active with half-maximal inhibitory concentrations (IC₅₀s) of 0.63 and 1.11 µg/mL against the 2 strains, respectively (Table 1). ¹⁶ In fact, Pamianthe peruviana stood out as the most active of the 19 plants against the resistant K1 strain with an IC₅₀ nearly 3 times that of the second most active plant Combretum hartmannianum Schweinf. (Combretaceae) (IC₅₀ 1.52 µg/mL). ¹⁶ The activity of Pamianthe peruviana was also significant against the sensitive NF54 strain (IC₅₀ 1.11 µg/mL) wherein IC₅₀s ranged from 0.2 µg/mL (Combretum hartmannianum) to 3.56 µg/mL (Euphorbia thi Schweinf., Euphorbiaceae). ¹⁶ In spite of these good activities, IC₅₀s determined for the chloroquine standard were 0.09 and 0.004 µg/mL against the K1 and NF54 strains, respectively. ¹⁶ These findings are, thus, congruent with the traditional usage of Pamianthe peruviana in Sudanese medicinal culture. ¹⁶

The other species of Crinum investigated for antiplasmodial effects include C. asiaticum, C. bulbispermum, C. erubescens, and C. macowanii. ^{17,23 -25} An extensive survey by Clarkson et al identified approximately 700 plant taxa from the southern African region, which have applications in ethnic medicine for the treatment of malaria. ¹⁷ A screen against the chloroquine-sensitive D10 strain of Plasmodium falciparum was subsequently carried out on 134 species, of which 66 exhibited promising activities (IC₅₀s ≤ 10 µg/mL), while 23 others were found to be highly active (IC₅₀s ≤ 5 µg/mL). ¹⁷ The single Amaryllidaceae representative C. macowanii was shown to have moderate activity as its water and DCM/MeOH bulb extracts exhibited IC₅₀ values of 25 and 26 µg/mL, respectively (Table 1). ¹⁷ Griffiths carried out a study on another South African Amaryllidaceae medicinal plant, ²³ in this instance the “Orange River lily” C. bulbispermum motivated by its reputed antimalarial properties in TM. ^{13 -15} Here, antimalarial activity was gaged by the [³H]-hypoxanthine incorporation assay utilizing the chloroquine-resistant Gambian FCR-3 strain of Plasmodium falciparum. ²³ In this way it was demonstrated that DCM and EtOAc bulb extracts had excellent activities with IC₅₀ values of 0.38 and 0.08 µg/mL, respectively. ²³ Subsequent phytochemical analysis identified lycorine (1) as the compound responsible for these antiplasmodial effects, with an IC₅₀ value of 0.029 µg/mL against the FCR-3 strain. ²³ By contrast, the IC₅₀ value determined for quinine in the same instance was 0.13 µg/mL. ²³ As part of a study probing the antimalarial potential of plants native to Costa Rica, C. erubescens was examined for antiplasmodial effects against the chloroquine/mefloquine-resistant Plasmodium falciparum strain Dd2. ²⁴ The initial methanol extract of C. erubescens was partitioned between aqueous methanol and hexane from which the methanol was removed, dried, and resuspended in water. ²⁴ An ethyl acetate extraction of the water phase afforded a fraction that proved to be active against the Dd2 strain (IC₅₀ <1.25 µg/mL). ²⁴ The artemisinin standard in the same instance reflected an IC₅₀ of 0.002 µg/mL. ²⁴ The follow-up phytochemical examination revealed that the cripowellin structures (3 and 4) were the most active antiplasmodial compounds present in the plant with IC₅₀ values of 0.016 and 0.014 µg/mL, respectively. ²⁴ C. asiaticum, as mentioned above, is known in Asian TM for its uses against malaria. ¹⁸ This formed the basis for a recent study that examined the in vitro antiplasmodial activities of this plant against both choroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum. ²⁵ The results showed that its chloroform bulb extracts were indeed active against the resistant RKL-​2 as well as the sensitive 3D-​7 strains (IC₅₀s 5.78 and 3.18 µg/mL, respectively). ²⁵ The test standard chloroquine exhibited IC₅₀s of 0.15 and 0.02 µg/mL against these strains, respectively. ²⁵ Interestingly, hemolytic assays of the extracts revealed that they exhibited minimal toxicity on mammalian red blood cells. ²⁵ Furthermore, subacute oral toxicity analyses indicated that extracts of C. asiaticum were well tolerated by Swiss albino mice even at concentrations as high as 2000 mg/g bdw. ²⁵ Thus, the use of the 4 Crinum species C. asiaticum, C. bulbispermum, C. erubescens, and C. macowanii in traditional malarial treatments has been corroborated by convincing in vitro antiplasmodial activities. ^{17,23 -25}

The other species examined for antiplasmodial effects include the African representatives Amaryllis belladonna Steud., Brunsvigia littoralis R.A.Dyer and Narcissus broussonetii Lag., as well as the Turkish members Leucojum aestivum L., Narcissus tazetta L., and Pancratium maritimum L.. ^{26 -29} Although Brunsvigia littoralis is not known in South African TM for uses against malaria, its ethanolic bulb extracts were suggested to be active against the D10 and FAC8 Plasmodium falciparum strains, which represent sensitivity and resistance to chloroquine, respectively. ²⁶ The phytochemical component of the work was insightful in identifying lycorine (1) and 1,2-O-diacetyllycorine (5) as the resident antiplasmodial entities of the plant. ²⁶ To this extent, lycorine exhibited IC₅₀s of 0.62 and 0.70 µg/mL against strains D10 and FAC8, respectively. ²⁶ 1,2-O-Diacetyllycorine (5), on the other hand, reflected IC₅₀ values of 1.0 µg/mL in both strains. ²⁶ These measurements were made against the artemisinin reference standard, which exhibited potent activities against both strains (IC₅₀s 0.004 and 0.02 µg/mL, respectively). ²⁶ Similarly, A. belladonna has no traditional value when it comes to treating malaria in South Africa although its methanolic bulb extracts did in fact reflect significant activities against the chloroquine-resistant strain Dd2. ²⁷ The accompanying screen utilizing alkaloids isolated from the bulbs highlighted acetylcaranine (6) and ambelline (7) (Figure 1) as the compounds possibly responsible for these effects. ²⁷ Acetylcaranine (IC₅₀ 1.1 µg/mL) proved to be twice as active as ambelline (IC₅₀ 2.42 µg/mL) against the Dd2 strain of Plasmodium falciparum. ²⁷ The Moroccan Amaryllidaceae plant N. broussonetii was examined in the assay against both the K1 and NF54 strains of Plasmodium falciparum, where EtOAc bulb extracts in each case were inactive. ²⁸ Şener et al then examined ethanolic extracts of the 3 Turkish medicinal plants L. aestivum, N. tazetta, and Pancratium maritimum for toxic effects on 2 strains of Plasmodium falciparum, including the chloroquine-resistant K1 strain as well as the chloroquine-sensitive T9.96 strain. ²⁹ In this case, all 3 plants were shown to be active in the T9.96 cultures with IC₅₀s of 1.47, 2.55, and 1.56 µg/mL, respectively. ²⁹ Similarly, IC₅₀s of 1.75, 3.31, and 1.83 µg/mL, respectively, were determined for the 3 plants against the K1 strain. ²⁹ The standards chloroquine and mefloquine exhibited IC₅₀s of 6.06 and 3.94 µg/mL in the K1 strain. ²⁹ Although several alkaloids were described from these plants, it was the crinane analogs haemanthamine (8) and haemanthidine (9), which proved to be the most efficacious across both these strains. ²⁹ As such, IC₅₀ values seen against the T9.96 strain were 0.70 and 0.35 µg/mL for both compounds, respectively. ²⁹ Activities against the K1 strain were even more impressive since haemanthamine and haemanthidine exhibited IC₅₀ values of 0.43 and 0.35 µg/mL, respectively. ²⁹

In summary, this survey attests to the significant number of studies that have been carried out to verify the antiplasmodial effects of the Amaryllidaceae via its plant extracts. To date, 18 plants from 11 genera of the family have been screened against 2 species of Plasmodium, including the avian parasite Plasmodium gallinaceum and the human parasite Plasmodium falciparum. Crinum with 5 species was the most studied genus. The extracts that were prepared mostly from organic solvents were screened against 11 different strains of the malarial parasite Plasmodium falciparum, including both choroquine-sensitive and chloroquine-resistant strains. The best overall activity was observed for an EtOAc extract of C. bulbispermum against the FCR-3 strain (IC₅₀ 0.08 µg/mL). The screen against Plasmodium gallinaceum was conducted in vivo and the best activity here was an MED of 3.2 mg/kg involving an EtOH extract of Z. drummondii. Interestingly, 6 of the 11 Amaryllidaceae species documented in TM for their usage against malaria have all responded positively in assays of the plasmodial parasite, with C. bulbispermum, C. macowanii, and Pamianthe peruviana standing out for their potent activities. These activities were shown to reside with the isoquinoline alkaloid principles of the family, some of which such as lycorine, haemanthamine, and haemanthidine were operative at the submicromolar level. Future studies will undoubtedly continue to focus on those species that have a traditional relevance in the remediation of malaria.