Abstract
Context: Malaria is one of the most common and serious protozoan tropical diseases. Multi-drug resistance remains pervasive, necessitating the continuous development of new antimalarial agents.
Objective: Many glycosides, such as triterpenoid saponins, were shown to have antimalarial activity against Plasmodium falciparum in vitro. This study was to elucidate the ability of five glycoalkaloids against Plasmodium yoelii and develop new antimalarial lead compounds.
Materials and methods: Glycoalkaloids were isolated from three kinds of Solanaceae plants: chaconine and solanine were isolated from Solanum tuberosum L. sprouts, solamargine and solasonine from Solanum nigrum L. fruit, tomatine from Lycopersicon esculentum Mill. fruit. The five isolated glycoalkaloids were evaluated against Plasmodium yoelii 17XL in mice with 4-day parasitemia suppression test in different concentrations.
Results: Chaconine showed a dose-dependent suppression of malaria infection, ED50, 4.49 mg/kg; therapeutic index (TI), ≈9. At a dose of 7.50 mg/kg, the parasitemia suppressions of chaconine, tomatine, solamargine, solasonine and solanine were 71.38, 65.25, 64.89, 57.47 and 41.30%, respectively. At 3.75 mg/kg, the parasitemia suppression of chaconine was 42.66%, but the derivative, chaconine-6-O-sulfate, appeared to show no antimalarial activity. Simultaneous administration of chaconine and solanine in 1:1 did not show any synergistic effects.
Discussion and conclusion: The results showed that the glycoalkaloids with chacotriose (chaconine and solamargine) were more active than those with solatriose (solanine and solasonine). Chaconine was the most active among the five glycoalkaloids. We propose that the activity is dependent upon non-specific carbohydrate interactions. The 6-OH of chaconine is important for antimalarial activity.
Introduction
Malaria is one of the most common and serious protozoan tropical diseases, and is transmitted to humans by mosquitoes. Considerable success in malaria prevention and cure has been achieved with unremitting efforts (CitationWyler, 1993; CitationAlecrim et al., 1999; CitationKshirsagar et al., 2000; CitationWhite, 2004). However, multi-drug resistance remains pervasive, necessitating the continuous development of new antimalarial agents.
Glycosides, such as triterpenoid saponins, flavonol glycosides, pregnane glycosides, stilbene glycosides, and resin glycosides (CitationTraore et al., 2000; CitationSchmieg et al., 2003; CitationLiu et al., 2007; CitationMontenegro et al., 2007; CitationAbdel-Sattar et al., 2008; CitationPark et al., 2008; CitationTasdemir et al., 2008), have been reported to possess antimalarial activities. Glycoalkaloids are a group of glycosides found predominantly in the plants of the Solanaceae and the Liliaceae families. Plants are thought to produce glycoalkaloids to protect themselves from attacks from microbes, insects and animals. Previous studies have shown that glycoalkaloids are biologically active as antifungals (CitationFewell & Roddick, 1993), antimicrobials (CitationWanyonyi et al., 2003), antivirals (CitationThorne et al., 1985), cancer chemotherapeutics (CitationKuo et al., 2000; CitationLee et al., 2004; CitationFriedman et al., 2005), and embryotoxins (CitationFriedman et al., 1991).
To date, no study has investigated for antimalarial activities of glycoalkaloids. In the present study, chaconine, solanine solamargine, solasonine, tomatidine and chaconine-6-O-sulfate are shown to possess antimalarial activities. Structure-activity relationships and possible mechanisms of action of the glycoalkaloids are discussed.
Materials and methods
Isolation of glycoalkaloids and preparation of their derivatives
The glycoalkaloids were isolated from the Solanaceae plants as previously described by CitationFriedman et al. (1991). Chaconine and solanine were isolated from Solanum tuberosum L. sprouts, solasonine and solamargine were from Solanum nigrum L. fruit, and tomatine was obtained from Lycopersicon esculentum Mill. fruit. The compounds were purified by silica gel column chromatography and characterized by carbon 13 nuclear magnetic resonance (13C NMR). The purity of compounds (>98%) was confirmed by HPLC. The 6-OH of the d-glucose in chacotriose of chaconine was sulfated by chlorosulfonic acid in pyridine as described by CitationZhao et al. (2006).
Preparation of test solutions
Artesunate was purchased from Guiling Pharmaceutical (Guangxi, China). Before animal experimentation, all compounds were dissolved in approximately 1 mL 0.1 M HCl and brought to final volume with distilled water. The pH was adjusted to 6.8 with 1 M NaOH to make the stock solution. The various doses administered to the animals were prepared by serial dilution from the stock solution.
Animals and parasites
Out-bred female ICR mice, 4–6 weeks old, 20 ± 2 g body weight, were purchased from the Center for Pilot Animal of Jilin University. They were supplied ad libitum with a standard diet and tap water at a temperature of 22° ± 3°C. All procedures conducted by the Institute for Experimental Animals of Jilin University were carried out in strict accordance with the PR China legislation on the use and care of laboratory animals and were approved by the university committee for animal experiments.
The Plasmodium yoelii strain 17XL was a gift from Cao Ya-Ming (Department of Immunology, China Medical University) and blood stage parasites were stored in liquid nitrogen.
Methods for in vivo suppression of parasitemia
We employed a 4-day blood schizonticidal test modified from previous studies (CitationPeters et al., 1975; CitationElufioye & Agbedahunsi, 2004). Each mouse received standard inoculum of 1 × 107 Plasmodium yoelii-infected erythrocytes intraperitoneally on day 1. The test dose was administered once per day for 4 days. The vehicle of 10 mL/kg was administered to negative control mice intraperitoneally (i.p.), with the first dosing 3 h after infection. Artesunate was administered orally for 4 days as positive control (28 mg/kg per day). On day 5, thin blood films were made from mouse tail blood and stained by Giemsa. The films were examined using a microscope to assess the activities of the compounds. Percentage parasitemia in each field was calculated as:
where PRBC are parasitized red blood cells and RBC are red blood cells.
Percentage suppression was calculated as:
Antimalarial activity of chaconine and acute toxicity study
The dose response of the chaconine test was carried out on mice. The mice were divided into seven groups of ten mice each: five groups for chaconine testing, two for control. The groups were treated i.p. from day 1 to day 4 with increasing doses of chaconine ranging from 0.97 to 7.50 mg/kg. ED50 (dose at which 50% reduction of parasitemia is achieved) values were determined by linear interpolation from the inhibition curve.
The LD50 (50% lethal dose) of chaconine was determined using ICR mice by intraperitoneal administration according to CitationLorke (1983). Groups of ten mice received intraperitoneally doses of 20-60 mg/kg of chaconine, while the control group received only the vehicle. The groups were observed for 4 days and at the end of this period mortality was recorded for each group.
Antimalarial activity of chaconine-related glycoalkaloids
Evaluation of antimalarial activities of five individual glycoalkaloids was employed on mice. The mice were divided into seven groups of ten mice each: five for glycoalkaloid administration, two for controls. The test groups were given chaconine, solanine, solasonine, solamargine and tomatine at a single dose of 7.50 mg/kg/day.
Antimalarial activity of chaconine vs. 6-O-sulfated chaconine
The antimalarial activity of chaconine-6-O-sulfate was evaluated on mice, divided into four groups of ten mice each. Chaconine and chaconine-6-O-sulfate groups were administered at a dose of 3.75 mg/kg/day.
Antimalarial activity of co-administered chaconine and solanine
The evaluation of antimalarial activity of chaconine and solanine in combination was carried out on mice. The mice were divided into seven groups of ten mice each for treatment, positive control and negative control. Four treated groups were given chaconine and solanine individually, using doses of 3.75 and 7.50 mg/kg/day. The final treatment group was given 7.50 mg/kg/day each of chaconine and solanine in combination.
Statistical method
Results were expressed as the mean ± SD of the indicated number of experiments. For comparison of suppression of parasitemia, one-way ANOVA and Student’s t-test were used. Values of p <0.05 and p <0.01 were considered to be significant.
Results
The antimalarial activity of chaconine was tested at varying dosages during a 4-day suppression test. summarized the dose-dependent suppression of malaria infection by chaconine. At 4.50 and 7.50 mg/kg, chaconine exhibited significant (p <0.01) antimalarial activity. The ED50 of chaconine was 4.49 mg/kg/day.
Table 1. Antimalarial activity of chaconine over a 4-day period.
To evaluate the viability of chaconine as an antimalarial drug, acute toxicity was tested. Chaconine (20–60 mg/kg) caused physical signs of toxicity such as hyperpnea, lethargy and death depending on the dose. All the mice treated with doses of chaconine greater than 47.42 mg/kg died. The intraperitoneal LD50 of chaconine in mice was calculated to be 40.23 mg/kg. The therapeutic index (TI; ratio of LD50 to ED50) of chaconine by i.p. administration was 9.
Glycoalkaloids solanine, solamargine, solasonine and tomatine possessed similar structures to chaconine (). To compare the antimalarial activities and elucidate the structure-activity relationships of the isolated compounds, the related five glycoalkaloids were tested at 7.5 mg/kg/day using a 4-day parasitemia suppression test as employed for evaluation of chaconine. All five glycoalkaloids were found to have appreciable antimalarial activities at the dose of 7.5 mg/kg/day () with chaconine being the most active (suppression: 71.38%). The levels of suppression of parasitemia exhibited by tomatine, solamargine, solasonine and solanine were 65.25, 64.89, 57.47 and 41.30%, respectively.
Figure 1. Structures of glycoalkaloids.
Table 2. Antimalarial activity of chaconine and related glycoalkaloids over a 4-day period.
To better define the role of its carbohydrate moiety, chaconine was selectively sulfated at the O-6 position of the carbohydrate chain according to the previously described methods in our laboratory (CitationZhao et al., 2006), and its antimalarial activity was determined. Chaconine-6-O-sulfate possessed no antimalarial activity at a dose of 3.75 mg/kg/day (). However, at the same dose, the suppression of parasitemia by chaconine was 42.66%.
Table 3. Antimalarial activity of chaconine versus 6-O-sulfated chaconine over a 4-day period.
To examine potential synergistic effects of co-administration of glycoalkaloids (), we investigated the antimalarial effects of chaconine and solanine both individually and in combination (1:1). Co-administration of solanine and chaconine (3.75 mg/kg/day of each compound) resulted in 60.75% suppression of parasitemia, which was less than the sum of suppression by chaconine and solanine individually (chaconine, 42.66% suppression at 3.75 mg/kg/day; solanine, 30.01% suppression at 3.75 mg/kg/day). However, compared with suppression of parasitemia by chaconine and solanine individually at 7.50 mg/kg/day, the combination showed weaker activity than chaconine alone and stronger activity than solanine alone. These results suggest that chaconine and solanine do not act synergistically.
Figure 2. The antimalarial (in vivo) activities of solanine and chaconine individually and in combination (1: 1). S, solanine; C, chaconine; 3.75, 7.5 represent concentration (mg/kg/day).
Discussion
Chaconine, solanine, solamargine, solasonine and tomatine are the predominant glycoalkaloids produced by the Solanaceae plant family. Glycoalkaloids consist of a nitrogen-containing steroid (aglycone) and a carbohydrate side chain. Chaconine and solanine are based upon the aglycone, solanidine; and solamargine and solasonine are based upon the aglycone, solasodine. The carbohydrate chains of chaconine and solamargine are the branched bis(α-l-rhamnopyranosyl)-β-d-glucopyranose known as chacotriose, and the carbohydrate chains of solanine and solasonine are the branched α-l-rhamnopyranosyl-β-d-glucopyranosyl-β-d-galactopyranose known as solatriose. Parasitemia suppression (at 7.5 mg/kg/day) by chaconine (71.38%) is higher than that of solanine (41.3%), while solamargine (64.89%) is higher than that of solasonine (57.47%). The results indicated that the glycoalkaloids with chacotriose exhibited greater suppression than the glycoalkaloids with solatriose against Plasmodium yoelii in mice.
Our results were similar to those previously reported for the glycoalkaloids chaconine and solanine in disrupting cell membranes (CitationKeukens et al., 1995, Citation1996), antifungal activity (CitationFewell & Roddick, 1993), blocking of sodium ion active transport in frog skin (CitationBlankemeyer et al., 1995), antitumorigenic activity (CitationLee et al., 2004; CitationFriedman et al., 2005) and Xenopus embryo teratogenesis (CitationFriedman et al., 1992; CitationRayburn et al., 1994). As mentioned above, the correlation of solamargine and solasonine in antimalarial activities was similar to that in antifungal activity (CitationCipollini & Levey, 1997), developmental toxicology in frog embryos (CitationBlankemeyer et al., 1998) and antiproliferative activities against human colon and liver cancer cell lines (CitationLee et al., 2004).
A variety of glycosides have been investigated for potential antimalarial activities (CitationTraore et al., 2000; CitationSchmieg et al., 2003; CitationLiu et al., 2007; CitationMontenegro et al., 2007; CitationAbdel-Sattar et al., 2008; CitationLee et al., 2008; CitationPark et al., 2008; CitationTasdemir et al., 2008), and mechanistic studies have focused predominantly on the nature of cell membrane disruption by glycosides. The resin glycosides cryptophilic acids 1 and 3 interfered with and disrupted glycolipid biosynthesis on the cell surface of the parasite, and exhibited antimalarial activity (CitationTasdemir et al., 2008). The toxicity of steroidal saponins isolated from Yucca schidigera Roezl. toward protozoans appears widespread and non-specific, and results from detergent effects on the cell membranes (CitationWang et al., 2000). It was also reported that solamargine and solasonine inhibited Trypanosoma cruzi (the causative agent of Chagas disease) by cytolysis, which was independent of rhamnose receptor-specific interactions (CitationHall et al., 2006). We therefore suggest that the antimalarial activities of glycoalkaloids are dependent upon non-specific carbohydrate interactions, which led to the formation and intercalation of sterol complexes into the plasma membrane of P. yoelii.
Tomatine has a tetrasaccharide chain attached to the aglycone tomatidine. Tomatine appears to suppress parasitemia more potently than the other glycoalkaloids, save for chaconine. This may be attributed to the ability of tomatine to disrupt cell membranes (CitationLee et al., 2004) and to enhance the immune response via induction of cytokines in immunized animals (CitationHeal et al., 2001).
It has been found that chaconine is more active than solamargine, solasonine, solanine and tomatine against human colon and liver cancer cells (CitationLee et al., 2004). Similarly, in our tests of suppression of Plasmodium yoelii parasitemia in mice, chaconine is most active among the five glycoalkaloids. The activities of glycosides can be altered by modification of sugar chains. The cytotoxicity of chaconine and solanine against frog embryos can be modulated by varying sugar chain identity (CitationRoddick & Rijnenberg, 1986). In the present study, we sulfated chaconine at C-6 position to produce chaconine-6-O-sulfate. Surprisingly, chaconine lost all activity against Plasmodium yoelii in mice after sulfation, demonstrating the importance of the 6-OH for activity.
Some Solanum species produce two predominant glycoalkaloids, termed “paired glycoalkaloids”. Potatoes produce glycoalkaloids chaconine and solanine, while black nightshade (Solanum nigrum) produces glycoalkaloids solamargine and solasonine. Several studies have suggested that the paired glycoalkaloids might act synergistically in plant defense. Positive synergistic effects have been observed for chaconine and solanine in permeation of liposomes (CitationRoddick & Rijnenberg, 1986), disrupting membranes (CitationKeukens et al., 1995), antifungal (CitationFewell & Roddick, 1993) and antitumorigenic effects (CitationFriedman et al., 2005). However, combinations of chaconine and solanine were slightly antagonistic or non-interactive in acetylcholinesterase inhibition (CitationRoddick, 1989). In our study, co-administration of chaconine and solanine did not act synergistically to suppress Plasmodium yoelii parasitemia in mice. Significantly, most previous studies that reveal synergistic effects were examined in vitro. The positive results obtained in vitro may not be consistent with those obtained in vivo.
TI is a quantitative measurement of the relative safety of drugs. In this paper, the TI of chaconine is 9 by i.p. administration, implying that chaconine might be as a potential lead compound which could be modified to be a candidate for an antimalarial drug. However, we failed to increase the activity by sulfation at 6-OH of glucose. The related modification of chaconine is ongoing investigation in our research group.
In the present paper, the antimalarial activities of glycoalkaloids were tested using Plasmodium yoelii on mice. Previous results showed that the antimalarial activities of some natural products in vivo do not necessarily correlate with those in vitro (CitationGessler et al., 1995; CitationMuthaura et al., 2007). This may be due to biotransformation of the constituents or poor bioavailability of the compounds in vivo. In addition, the most common human pathogen is Plasmodium falciparum. Thus, further research on the antimalarial activity of chaconine would be the structural modification to find the suitable candidate compound. Then, the evaluation would be carried out both in vitro using Plasmodium falciparum and in vivo using Plasmodium yoelii. The mechanisms underlying the antimalarial activity of glycoalkaloids will require further investigation too.
In summary, five glycoalkaloids from Solanaceae plants were examined for antimalarial activity, and chaconine was found to be the most active. Our study is the first report of antimalarial activity of glycoalkaloids. The results suggest that glycoalkaloids may be potent new leads for the development of antimalarial agents.
Acknowledgements
The authors are grateful to Yaming Cao (China Medical University, Shenyang) for providing the strain of P. yoelii employed in the study and other technical assistances.
Declaration of interest
This work was supported by the Natural Science Foundation of Jilin Province (20040546) and Analysis and Testing Foundation of Northeast Normal University.
References
- Abdel-Sattar E, Harraz FM, Al-ansari SMA, El-Mekkawy S, Ichino C, Kiyohara H, Ishiyama A, Otoguro K, Omura S, Yamada H (2008). Acylated pregnane glycosides from Caralluma tuberculata and their antiparasitic activity. Phytochemistry 69: 2180–2186.
- Alecrim MDGC, Alecrim W, Macedo V (1999). Plasmodium vivax resistance to chloroquine (R2) and mefloquine (R3) in Brazilian Amazon region. Rev Soc Bras Med Trop 32: 67–68.
- Blankemeyer JT, Atherton R, Friedman M (1995). Effect of potato glycoalkaloids α-chaconine and α-solanine on sodium active transport in frog skin. J Agric Food Chem 43: 636–639.
- Blankemeyer JT, McWilliams ML, Rayburn JR, Weissenberg M, Friedman M (1998). Developmental toxicology of solamargine and solasonine glycoalkaloids in frog embryos. Food Chem Toxicol 36: 383–389.
- Cipollini ML, Levey DJ (1997). Antifungal activity of Solanum fruit glycoalkaloids: Implications for frugivory and seed dispersal. Ecology 78: 799–809.
- Elufioye TO, Agbedahunsi JM (2004). Antimalarial activities of Tithonia diversifolia (Asteraceae) and Crossopteryx febrifuga (Rubiaceae) on mice in vivo. J Ethnopharmacol 93: 167–171.
- Fewell AM, Roddick JG (1993). Interactive antifungal activity of the glycoalkaloids α-solanine and α-chaconine. Phytochemistry 33: 323–328.
- Friedman M, Lee KR, Kim HJ, Lee IS, Kozukue N (2005). Anticarcinogenic effects of glycoalkaloids from potatoes against human cervical, liver, lymphoma, and stomach cancer cells. J Agric Food Chem 53: 6162–6169.
- Friedman M, Rayburn JR, Bantle JA (1991). Developmental toxicology of potato alkaloids in the frog embryo teratogenesis assay – Xenopus (FETAX). Food Chem Toxicol 29: 537–547.
- Friedman M, Rayburn JR, Bantle JA (1992). Structural relationships and developmental toxicity of Solanum alkaloids in the frog embryo teratogenesis assay – Xenopus. J Agric Food Chem 40: 1617–1624.
- Gessler MC, Tanner M, Chollet J, Nkunya MHH, Heinrich M (1995). Tanzanian medicinal plants used traditionally for the treatment of malaria: In vivo antimalarial and in vitro cytotoxic activities. Phytother Res 9: 504–508.
- Hall CA, Hobby T, Cipollini M (2006). Efficacy and mechanisms of α-solasonine- and α-solamargine-induced cytolysis on two strains of Trypanosoma cruzi. J Chem Ecol 32: 2405–2416.
- Heal KG, Sheikh NA, Hollingdale MR, Morrow WJW, Taylor-Robinson AW (2001). Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a pre-erythrocytic malaria vaccine candidate antigen. Vaccine 19: 4153–4161.
- Roddick JG, Rijnenberg AL (1986). Effect of steroidal glycoalkaloids of the potato on the permeability of liposome membranes. Physiol Plant 68: 436–440.
- Keukens EAJ, De Vrije T, Jansen LAM, De Boer H, Janssen M, De Kroon AIPM, Jongen WMF, De Kruijff B (1996). Glycoalkaloids selectively permeabilize cholesterol containing biomembranes. Biochim Biophys Acta 1279: 243–250.
- Keukens EAJ, De Vrije T, Van den Boom C, De Waard P, Plasman HH, Thiel F, Chupin V, Jongen WMF, De Kruijff B (1995). Molecular basis of glycoalkaloid induced membrane disruption. Biochim Biophys Acta 1240: 216–228.
- Kshirsagar NA, Gogtay NJ, Rajgor D, Dalvi SS, Wakde M (2000). An unusual case of multidrug-resistant Plasmodium vivax malaria in Mumbai (Bombay), India. Ann Trop Med Parasitol 94: 189–190.
- Kuo KW, Hsu SH, Li YP, Lin WL, Liu LF, Chang LC, Lin CC, Lin CN, Sheu HM (2000). Anticancer activity evaluation of the Solanum glycoalkaloid solamargine: Triggering apoptosis in human hepatoma cells. Biochem Pharmacol 60: 1865–1873.
- Lee KR, Kozukue N, Han JS, Park JH, Chang EY, Baek EJ, Chang JS, Friedman M (2004). Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells. J Agric Food Chem 52: 2832–2839.
- Lee SI, Yang HD, Son IH, Moon HI (2008). Antimalarial activity of a stilbene glycoside from Pleuropterus ciliinervis. Ann Trop Med Parasitol 102: 181–184.
- Liu Y, Murakami N, Ji H, Abreu P, Zhang S (2007). Antimalarial flavonol glycosides from Euphorbia hirta. Pharm Biol 45: 278–281.
- Lorke D (1983). A new approach to practical acute toxicity test. Arch Toxicol 54: 275–286.
- Montenegro H, González J, Ortega-Barria E, Cubilla-Rios L (2007). Antiprotozoal activity of flavonoid glycosides isolated from Clidemia sericea and Mosquitoxylon jamaicense. Pharm Biol 45: 376–380.
- Muthaura CN, Rukunga GM, Chhabra SC, Omar SA, Guantai AN, Gathirwa JW, Tolo FM, Mwitari PG, Keter LK, Kirira PG, Kimani CW, Mungai GM, Njagi ENM (2007). Antimalarial activity of some plants traditionally used in treatment of malaria in Kwale district of Kenya. J Ethnopharmacol 112: 545–51.
- Park W, Lee S, Moon H (2008). Antimalarial activity of a new stilbene glycoside from Parthenocissus tricuspidata in mice. Antimicrob Agents Chemother 52: 3451–3453.
- Peters W, Portus JH, Robinson BL (1975). The chemotherapy of rodent malaria, XXII. the value of drug resistant strains of P. berghei in screening for blood schizontocidal activity. Ann Trop Med Parasitol 69: 155–171.
- Rayburn JR, Bantle JA, Friedman M (1994). Role of carbohydrate side chains of potato glycoalkaloids in developmental toxicity. J Agric Food Chem 42: 1511–1515.
- Roddick JG (1989). The acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycones. Phytochemistry 28: 2631–2634.
- Schmieg J, Yang G, Franck RW, Tsuji M (2003). Superior protection against malaria and melanoma metastases by a C-glycoside analogue of the natural killer T cell ligand α-galactosylceramide. J Exp Med 198: 1631–1641.
- Tasdemir D, Brun R, Franzblau SG, Sezgin Y, Çalis I (2008). Evaluation of antiprotozoal and antimycobacterial activities of the resin glycosides and the other metabolites of Scrophularia cryptophila. Phytomedicine 15: 209–215.
- Thorne HV, Clarke GF, Skuce R (1985). The inactivation of herpes simplex virus by some Solanaceae glycoalkaloids, Antiviral Res 5: 335–343.
- Traore F, Faure R, Ollivier E, Gasquet M, Azas N, Debrauwer L, Keita A, Timon-David P, Balansard G (2000). Structure and antiprotozoal activity of triterpenoid saponins from Glinus oppositifolius. Planta Med 66: 368–371.
- Wang Y, McAllister TA, Yanke LJ, Cheeke PR (2000). Effect of steroidal saponin from Yucca schidigera extract on ruminal microbes. J Appl Microbiol 88: 887–896.
- Wanyonyi AW, Chhabra SC, Mkoji G, Njue W, Tarus PK (2003). Molluscicidal and antimicrobial activity of Solanum aculeastrum. Fitoterapia 74: 298–301.
- White NJ (2004). Antimalarial drug resistance. J Clin Invest 113: 1084–1092.
- Wyler DJ (1993). Malaria: Overview and update. Clin Infect Dis 16: 449–458.
- Zhao JM, Li SY, Zhou YF, Zhang LP, Zhou DW (2006). 6-O-Sulfated modification of natural glycoalkaloids chaconine and solanine. Chem Res Chinese Univ 22: 189–192.