Development of the drug resistant Plasmodium and insecticide resistant vector strains has led to the difficulty in eradication and control of malaria. According to the World Malaria Report 2011, there were 216 million cases and an estimated 6,55,000 persons died of malaria in 2010, and 91% of malaria deaths occurred in the WHO African Region . The present study has been undertaken to explore what homeopathy could offer, both for cure as well as prophylaxis. Homeopathic drugs have been claimed for their efficacy without inflicting side effects. It is a therapeutic system that uses small doses of natural substances to stimulate autoregulatory and self-healing process. Attempts have also been made to explain the mechanism of biological responses to homeopathic drugs, by conducting both in vitro and in vivo experiments . Homeopathic medicines, during the dynamization/potentization process, interact with nano-particles that eventually may have some role in tagging on to some proteins that eventually act as ligand which binds to receptors to elicit specific response . Well-selected homeopathic remedies accelerate cellular and sub-cellular activities that are expected to be associated with the recovery process . But, there is a lack of conclusive scientific evidence on the effectiveness of homeopathy for most conditions.
For homeopathic research, mice (Mus musculus) have been used as experimental models as they share a high degree of homology with humans . Salazar et al.  demonstrated the effect of homeopathic medicines Eupatorium perfoliatum 30CH and Arsenicum album 30CH on Plasmodium berghei infected mice. Cinchona officinalis (china) mother tincture has also been reported as an efficient antimalarial in monotherapy and a partner drug for artesunate-based combination therapy against P. berghei infection in BALB/c mice . China 30C has been reported to exhibit 100% chemosuppression with enhanced MST of mice. It also showed hepatoprotective and nephroprotective activity against detrimental fate of liver and kidney of mice infected with P. berghei . China has also been reported to cure low forms of remittent or intermittent, or malarial fever and also kills parasites . It is likely that both Cinchona and malaria share some common effector toxin resulting in a similar illness. Homeopathic drug Cinchona neutralizes this common effector toxin which leads to the cure of malaria .
Another homeopathic preparation Chelidonium majus 30C is reported to exhibit significant antiplasmodial effect against lethal rodent malaria parasite and, also enhanced the survival time of mice as compared to its mother tincture and 6/200 potencies . Extracts of Chelidonium majus are traditionally used in various complementary and alternative medicine (CAM) systems including homeopathy, mainly in combating diseases of the liver  Homeopathic formulation of Chel. 3X is also recommended for annually recurring type of malaria . The present study has been undertaken to evaluate antimalarial potential of Cinchona officinalis (Chin.) 30C and Chelidonium majus (Chel.) 30C in combination therapy against lethal rodent malaria parasite.
Materials and methods
Mouse and parasite strain
White swiss mice (4–6 weeks old) Mus musculus of BALB/c strain (weighing 22–25 g) and of either sex were used as an experimental model. These were obtained from the Central animal house, Panjab University, Chandigarh. Animals were maintained on a standard pellet diet and water ad libitum. Chloroquine sensitive P. berghei (NK-65) was maintained by i.p. inoculation of 1×106 infected RBCs to naïve mice. Parasitaemia was checked by preparing Giemsa stained thin blood smears through tail vein incision of infected mice . The experimental protocol was approved by the ethical committee (Reg No. 45/1999/CPCSEA), Panjab University, Chandigarh, India.
Homeopathic medicines (Chin. 30C and Chel. 30C) manufactured by Dr. Reckeweg and Co. GmbH D.64625, Bensheim, Germany, were used in the present study. Absolute alcohol (vehicle for homeopathic preparations) was given as placebo control. The medicines were diluted in distilled water (1:2).
Evaluation of schizonticidal activity in early infection (Peter’s 4-day test)
Five groups having 12 mice each (same sex and age) were used for the experiment (Table 1). The schizonticidal activity was assessed by the method described by Knight and Peters . 0.2 ml/mouse/day of drug/vehicle was administered orally, for four consecutive days (D0–D3). Infected control group was administered with distilled water. Artesunate (AS), sulphadoxine and pyrimethamine (SP) IP Combikit (FALCIGO-SP KIT, manufactured in India) containing separate tablets of artesunate with 200 mg base, sulphadoxine with 750 mg base and pyrimethamine with 37.5 mg base were used as positive control. AS was dissolved in 1 ml 5%Na2HCO3 and 5 ml 0.9%NaCl whereas, SP was dissolved in distilled water to make the required concentration and the dose of AS (4 mg/kg, OD for 3 days D0-D2)+SP (1.2 mg/kg, single dose on D3) was administered orally.
On the fifth day (D4), thin blood smears were prepared. Mice were kept for follow-up studies up to 4 weeks, including the dose regimen to calculate the parasitaemia, chemosuppression and mean survival time (MST).
Evaluation of the preventive activity
The preventive (repository) activity was assessed using the method described by Peters . Three groups (n=12) were administered with combination of Chin. 30C+Chel. 30C, pyrimethamine (1.2 mg/kg) and distilled water, for four consecutive days (D0–D3). On day 5 (D-4), mice were inoculated with 1×106 P. berghei infected erythrocytes. Seventy-two hours later i.e. D7, the parasitaemia level was assessed by studying Giemsa-stained blood smears. The MST (days) in each group was determined over a period of 28 days.
Evaluation of curative activity of homeopathic drugs in established infection
A modified method similar to that described by Ryley and Peters  was employed to evaluate the curative activity of the experimental drugs in established infection of P. berghei. On D0, all mice were injected (i.p.) with standard inoculum of 1×106 P. berghei-infected erythrocytes. Seventy-two hours later, mice were administered various drugs. Chloroquine (20 mg/kg/day for 5 days OD) was given to the positive control group. Parasitaemia levels were monitored for 5 days from day 3 (D3) onwards. The MST (days) for each group was determined over a period of 28 days.
Data has been presented as Mean±SD. Statistical evaluation of differences between the experimental groups was determined by the Student’s t-test with the level of significance of p<0.05. Data were subjected to analysis of variance (one-way ANOVA). Kaplan–Meier survival analysis was also done to illustrate the relative influence of the interventions. All the experiments were repeated for the validation of results.
Schizonticidal activity of drugs in early infection
The parasitaemia in infected control group increased up to 51.9±6% by day 7 leading to the death of all the mice due to heavy infection except for animals (n=4) in which parasite migrated to reticulocytes. These mice died within 2 weeks of infection (Table 1). The MST of this group was observed to be the lowest i.e. 7.6±0.4 days. Similar course of infection was observed in vehicle treated group. When Chin. 30C and Chel. 30C were given in combination, a slow increase in the infection was observed from day 7 i.e. 3.6±1.2% (p<0.0005) till day 14 (5.5±2.4%). After second week, a continuous decline in the parasitaemia was observed and infection was completely cleared with 100% parasite suppression by day 28 in 67% of mice. The MST of mice in this group was observed to be 26.8±1.2 days which was comparable to the positive control group (AS+SP) i.e. 28±0 days, respectively.
Extremely statistically significant (p<0.0005) antiplasmodial efficacy was observed in group treated with standard combination of AS+SP, in which the infection was completely cleared after day 7 with 100% chemosuppression. The MST of the group was found to be maximum i.e. 28±0 days. Difference between the parasitaemia of treated groups including the positive control AS+SP on day 7 was also found to be significant (p<0.0005) as observed in one-way ANOVA test. The survival difference between the experimental groups were observed to be significant with 95% confidence interval [Log Rank (Mantel-Cox) significance=0.000]. The Kaplan–Meier curve for survival analysis indicates all the groups as censored (Figure 1).
Preventive activity of various drugs
In the infected control, 7.45±2.1% of infection was observed on day 7 which increased to 26.5±4.7% by day 14 (Table 2). All the mice died after second week of study due to heavy infection. An extremely statistically significant (p<0.0005) reduction in infection (4.3±2.1%) was observed in pyrimethamine (1.2 mg/kg) treated group till day 14, and mice died after the third week post-inoculation (MST 21.8±1.47 days). In combination therapy group, the infection was 0.8±0.2% (p<0.0005) on day 7 followed by continuous increase in parasitaemia up to day 21 (23.6±4.22%).
The chemosuppression was 89.2% and the MST of the mice was recorded as 21.5±0.8 days which was extremely statistically significant (p<0.0005) when compared to infected control. The combination therapy was observed to possess significant preventive activity which was more than the preventive activity of pyrimethamine (83.8%).
The survival difference between the experimental groups were observed to be significant with 95% confidence interval [Log Rank (Mantel–Cox) significance=0.009]. The Kaplan–Meier curve for survival analysis indicates all the groups as censored (Figure 2).
Curative activity of Chin. 30C+Chel. 30C in combination therapy
There was a reduction in parasitaemia up to day 6 (0.28±0.1%, p<0.0005) in CQ (20 mg/kg) treated group. Parasitaemia was observed to increase slightly on day 14 (2.3±1%), but after the 3rd week of infection it was observed to decline up to 0.24±0.2% by day 28, and exhibited chemosuppressive activity of 99.1%, whereas the infected control group showed a daily increase in parasitaemia leading to the death of mice after day 7 (28.4±4.4%) (Table 3).
In the combination therapy group (Chin. 30C+Chel. 30C), the infection appeared on day 5 (1.02±0.1%) which remained almost similar till day 7 (1.24±1.04%, p<0.0005). After day 7, a continuous increase in parasitaemia was observed till day 21 i.e. 35.5±2.4%. The infection in these mice reduced to 20.7±3.91% by day 28. Four mice exhibited complete clearance of parasite from 3rd week with 100% chemosuppression. The MST was 26.8±2.8 days which was comparable to CQ treated group and was extremely statistically significant (p<0.0005) when compared to infected control.
The survival difference between the experimental groups were observed to be significant [Log Rank (Mantel–Cox) significance=0.000]. The Kaplan–Meier curve for survival analysis indicates all the three groups as censored (Figure 3).
The present study has been designed to evaluate the antiplasmodial efficacy of Chin. 30C and Chel. 30C in combination therapy with conclusive scientific evidence.
In earlier studies, monotherapy of Chin. 30C have been found to exert 100% chemosuppression, whereas Chel. 30C was reported to exhibit 93.8% chemosuppression against P. berghei infection in BALB/c mice [7, 10]. Hahnemann also theorized that any disease may remain unaffected by the lowest potency, whereas, higher potency probably is too powerful which may cause adverse effect, so the use of correctly selected medium potency may prove more effective . Therefore, 30C potency of Chin. and Chel. were selected for evaluating their schizonticidal, preventive and curative activities in combination therapy.
The combination exhibited extremely significant reduction in parasite load with 100% chemosuppression by the 28th day pointing to strong antimalarial efficacy of the combination. The MST of the combination Chin. 30C+Chel. 30C was also higher (26.8±1.2 days) and comparable to AS+SP treated group. In a laboratory study, combination of homeopathic medicine Eucalyptus and artesunate has been reported to provide mutual protection against blood stage infection of P. berghei in BALB/c mice. It also protected recrudescence up to 1 month follow-up period . In another study, the oral administration of Chin. (ϕ) led to inhibition of parasite load in P. berghei-infected Balb/C mice, with a 50% survival rate of mice till 1 month follow-up period. Combination therapy of artesunate (100 mg/kg) and Chin. ϕ was also found to be highly effective and it also enhanced the mean survival time of mice. The combination therapy was reported to be safe on structural arrangement of blood cells and pathophysiological state of liver and kidney of host [6, 19].
Biochemical assays were performed to assess the liver and kidney function tests of mice in the treated groups on day 7 and day 28. For the assessment of liver function tests, serum levels of SGOT, SGPT, ALP, and bilirubin have been carried out. While serum level of urea, creatinine, uric acid, and blood urea nitrogen have been used as a measure for assessment of renal sufficiency. The liver and kidney function tests in the Chin. 30C and Chel. 30C treated group was extremely statistically significant (p<0.0005) as compared to infected control. Histopathological studies of the liver and kidney were also carried out. The Chin. 30C and Chel. 30C treated liver showed normal morphology of liver except for the presence of Kupffer cell hyperplasia between the sinusoids. The histology of kidney revealed slight histopathological changes which include shrinkage of glomerular tufts and increased mesangial cell proliferation .
AS+SP was used as a positive control and 100% chemosuppression was observed with MST of 28±0 days which was maximum among all the treated groups. In another study by Georgewill and Ebong , AS+SP administration to P. berghei-infected mice was effective with mean percentage parasitaemia reduction of 100% by day 7. It reports complete clearance of parasites from the blood of treated mice, which is similar to the observations of the present study. In an open-double blind randomized clinical trial for malaria in Ghana, homeopathic remedies like Arsenicum album, china, Eup- perf., Nat- m., Nux- v., Pulsatilla, Rhus toxicodendron and Sulphur were used, and chloroquine was used as the standard drug in placebo control group. The percentage of improvement in open study – 90.7% and in double blind study – 83.3% has been attributed to the homeopathic treatment, during the 4-week study period . Considerable antiplasmodial efficacy of another homeopathic drug nosode 30 has been reported against lethal rodent malaria parasite. The safety of nosode 30 was also confirmed by the comparable levels of ALP, SGOT, SGPT activities, concentration of bilirubin, urea and creatinine to the positive control group treated with CQ (20 mg/kg) .
Davis et al.  reported that Cinchona interrupts schizogony in P. berghei. China is known to destroy the parasite by binding strongly to the blood proteins of the parasite . Different potencies of Cinchona officinalis is recommended for different pathological situations because the action of a remedy varies in relation to its potencies. China Q is more suitable for malarial poisoning, Chin. 3X or 6 is recommended for chronic diarrhoea, Chin. 30 or 200 for neuralgia and Chin. 1M or 10M for stimulating higher functions, immediately after the completion of an acute episode of malaria .
Chelidonium majus extracts are used mainly in the therapy of biliary and hepatic dysfunctions and it protects hepatotoxicity and shows an established therapeutic safety. They are in accordance with the available toxicity data for oral application, which show no hepatotoxic effects and give no indication for extended pharmacovigilance risk limitation . An immune stimulating effect of C. majus has also been noted .
During the preventive test, the chemosuppression was observed to be 89.2% in Chin. 30C+Chel. 30C treated group which was more than the positive control pyrimethamine (83.8%). It is also reported by Shah  that Cinchona officinalis can be taken as prophylactic drug in a 30C dose weekly prior to entering a malarial zone. On the other hand, the combination also exhibited strong curative efficacy. Chin. 30C+Chel. 30C also exhibited 100% chemossupressive activity in 50% of surviving mice, and also enhanced the MST of group up to 26.8±2.8 days in comparison to the infected control group in which the MST was 6.6±0.5 days. The positive control CQ (20 mg/kg) showed 99.1% chemo-suppressive activity on day 28 with MST of 28 days. In a curative experiment by Dow et al. , chloroquine reduced the mortality of mice in a dose-related fashion, and no mortality was observed in groups of mice treated with 6 mg/kg and 30 mg/kg of CQ. In another study, oral administration of CQ (20 mg/kg) once a day for four consecutive days showed complete recovery in 58.33% P. berghei-infected mice. These mice were alive till 28 days . A slight difference in the dose regimen may increase the preventive and curative activities of the experimental drugs and enhance the MST of treated mice.
Hahnemann noted that diluting and vigorously shaking homeopathic remedies (potentization) often rendered the remedy more potent in terms of clinical response which is once again confirmed in the present study. Considerable evidence indicates that the homeopathic remedies are capable of activating psycho-physiologic, self-regulatory mechanisms that mobilize hormones, neurotransmitters and components of immune system .
This study gives an insight about the use of homeopathy for the treatment of malaria, which will be effective in terms of cost and antimalarial efficacy without any drug pressure for resistance and side effects. These findings demonstrate that it is useful to combine drugs as is usually done in traditional methods. The mechanism of synergistic and additive interactions of the drugs could not be addressed in this study, so their interactions can be further investigated.
The authors are grateful to the PURSE programme of DST, New Delhi for financial support and the University Grants commission, New Delhi, India for the award of Rajiv Gandhi National fellowship to Aswathy Rajan.
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