WikiJournal Preprints/Inflammatory response in Plasmodium infected patients treated using raw liquid extract of Morinda lucida (Oowo) leaf in some traditional homes in Nigeria

Introduction
Morinda lucida is medicinal plant used for the treatment of Plasmodium infection in Nigeria owing to the phytochemical constituents and anti-oxidative activities[1][2]. Traditional medicine is becoming more acceptable in Nigeria because of the effectiveness[1][2].

In Nigeria the root extract is used to prepare alcohol beverages and the stem is used as chewing stick to clean mouth and teeth[1][2]. Raw liquid extract of the root, bark, and leaves have been used to treat fever, yellow fever, malaria, trypanosomiasis, and feverish condition during childbirth, diabetes, hypertension, cerebral congestion, dysentery, stomach ache, ulcers, leprosy, and gonorrhea[1][2].

The major phytochemicals in Morinda lucida include various types of alkaloids, anthraquinones, and anthraquinols. Oruwalol, oruwal and 10 anthraquinones have also been characterized in the stem extract. Generally the leaf extract of contains Steroids, Alkaloids, Terpenoids, Tannins, Saponins, Flavonoids, Phenols , hydrogen cyanide sand Glycosides[1][2][3][4][5][6].

Anthraquinones are naturally occurring phenolic compounds. The derivatives have been used as laxatives, antimicrobial, antiinflammatory agents, constipation, arthritis, multiple sclerosis, and cancer[7]. Plant steroids have health promoting bioactivities such as anti-tumor, immunosuppressive, hepatoprotective, antibacterial, plant growth hormone regulator, sex hormone, antihelminthic, cytotoxic and cardiotonic activity [8]. Plant alkaloids are group of phytochemicals used as antimalarial (e.g. quinine), antiasthma (e.g. ephedrine), anticancer (e.g. homoharringtonine),[9][10][11][12][13]. cholinomimetic (e.g. galantamine), [9][10][11][12][13] vasodilatory (e.g. vincamine), antiarrhythmic (e.g. quinidine), analgesic (e.g. morphine), [9][10][11][12][13] antibacterial (e.g. chelerythrine), [9][10][11][12][13] and antihyperglycemic activities (e.g. piperine). [9][10][11][12][13] psychotropic (e.g. psilocin) and stimulant activities (e.g. cocaine, caffeine, nicotine, theobromine), [9][10][11][12][13] and entheogenic as recreational drugs. Alkaloids can be toxic too (e.g. atropine, tubocurarine). [9][10][11][12][13].Plant terpenoids have substantial pharmacological including antioxidative bioactivities and are used in traditional herbal remedies[14][15][16]. Terpenoids include citral, menthol, camphor, salvinorin A,, cannabinoids, ginkgolide and bilobalide, and the curcuminoids and provitamin beta carotene which is a terpene derivative known as carotenoid[14][15][16]. Saponins a subclass of terpenoids have been used as adjuvants vaccines production but there is no high-quality clinical evidence that they have any beneficial effect on human health[17][18][19].

Plasmodium spp., is the causative agent of malaria fever. The protozoan is transmitted by female anopheles mosquito through blood inoculation[20][21]. The infection may lead to anaemia,electrolyte imbalance, hypoglycemia especially in children, kidney and liver dysfunction[20][21]. It is detectable in infected individuals through a thin and thick blood smear from the affected person[22].

Tumor necrosis factor alpha (TNF-α) that has a primary role in the regulation of immune cells is a pro-inflammatory cytokine released by the microphage upon infection to alert the cells as way of inflammatory response to stimulate phargocytosis.[23][24][25][26]. It acts as an endogenous pyrogen to induce fever, cause cell death, inhibit tumorogenesis and viral replication.[23][24][25][26]. Interleukin 10 (IL-10) or human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine which enhances B cell survival, proliferation, and antibody production[27][28]. It inhibits the production of pro-inflammatory cytokine and tumor metastasis[27][28].

This work was therefore designed to determine inflammatory response in Plasmodium infected patients treated using raw liquid extract of Morinda lucida leaf in some traditional homes in Nigeria

Study Area
The work was carried out in ten (10) traditional homes in Saki the headquarters of Saki-West local Government area in the northern part of Oyo State in the South Western part of Nigeria. There are three major hospitals in the area which include Oyo State Hospital Management Hospital, Saki, Baptist Medical Centre, Saki and Muslim Hospital Saki. Baptist Medical Centre, Saki provides primary, secondary and tertiary healthcare services.

Study Population
Fifty (50) Plasmodium infected participants(Female-25; Male-25; age- 14 – 64 years) receiving treatment were randomly recruited from the ten traditional homes; 50 age matched participants receiving malaria conventional treatments in the hospitals were recruited as test participants while 50 age matched non- Plasmodium infected participants were recruited as control. All participants were tested negative to AFB, HIV, HCV and HBV.

Biological samples
Sputum samples were obtained from each of the subjects in the cups provided for the detection of AFB. Venous blood preserved in lithium heparinized bottles was also obtained from each of the subjects for the identification and estimation of parasitaemia, HIVp24Ag-Ab ELISA, anti-HCVELISA, HBeAgELISA. TNFα and IL-10 ELISA

Preparation and Administration of raw liquid extract of Morinda lucida (Oowo)
The leaves of Morinda lucida were confirmed by the Oyo State Agricultural Development Programme, Saki, Nigeria. The preparation of the raw liquid extract was done by the traditional home practitioner and monitored by the researchers. Fresh leaves of the M. lucida were plucked and washed in water. Thereafter, the water was drained from the leaves. The leaves were then squished for the extraction of the liquid content into a sterile container. Seventy milliliter of the liquid extract using a 70ml capacity plastic cup was administered to the patients. The liquid extract was prepared and administered freshly on daily basis. The raw liquid content of the leaf is extracted without the addition of water and also administered undiluted.

HBeAg ELISA
Hepatitis B envelope antigen (HBeAg) was determined in test and control subjects by ELISA using the reagent kit of: DIA.PRO Diagnostic Bioprobes Srl Via Columella, Milano – Italy. Manufacturer’s instructions were strictly followed and applied.

HIVp24 antigen and antibodies ELISA
HIVp24 antigen and antibodies was determined in each of the subjects using Bio-Rad Genscreen™ ULTRA HIV Ag-Ab qualitative enzyme immunoassay kit Manufacturer’s instructions were strictly followed and applied.

Anti-HCV ELISA
Anti – HCV test was determined in each of the subjects by a third generation enzyme immunoassay reagent kit of DIA.PRO Diagnostic Bioprobes Srl Via Columella, Milano – Italy. Manufacturer’s instructions were strictly followed and applied.

TNF-α ELISA
Plasma TNF-α was determined in each of the subjects by ELISA. using Human ABCAM ELISA Kit. Manufacturer’s instructions were strictly followed and applied.

Plasmodium identification ad estimation of parasitaemia
Estimation of Plasmodium parasiteamia was carried out as described by Centers for Disease Control Prevention[29]. The Plasmodium was quantified against WBCs: on the thick smear. The parasite was tallied against WBCs, until 1,000 WBCs was counted and the results was expressed as parasites per microliter of blood, using the WBC count. Parasites/microliter blood = (parasites/WBCs) × WBC count per microliter.

Ziehl Nelseen Staining for Acid Fast Bacilli (AFB)
This was carried out as described by Cheesbrough[22].

Based on the information obtained from the 15 traditional homes visited in the three local governments about 70 ml of the raw undiluted liquid extract of the M. lucida leaf is administered to the patients on daily bases for at least 1 week.

Ethical Consideration
The proposal was reviewed and approved by the Research and Ethics Committee of Baptist Medical Centre, Saki, Nigeria before the commencement of the work.(BMC/IX/102) The consent of each subject was also obtained.

Statistical Analysis
The results of this work  was subjected to statistical analysis using SPSS 20.0 to determine mean, standard deviation, frequency Odd Ratio(OR), ANOVA, ‘t’ test and probability value at 0.05 level of significance.

Results
A comparative  analysis of plasma  IL-10 (pg/ml), TNF-α (pg/ml) and parasite density using ANOVA was carried out across the groups which include : Plasmodium infected  patients before treatment in traditional homes, Plasmodium infected patients before treatment in Hospitals, Plasmodium infected  patients after treatment in traditional homes, 	Plasmodium infected  patients after treatment ni Hospitals and Non- Plasmodium infected  Control. The results obtained showed a statistically significant difference in the  plasma IL-10 (pg/ml), TNF-α (pg/ml) and parasite density across the 5 groups(p<0.05; Table 1)

The plasma values of  IL-10 (pg/ml), TNF-α (pg/ml) and parasite density were also compared using student ‘ t ‘test to determine how each value varies with one group and another. The results obtained are therefore itemized below.


 * 1) There was a significant decrease in plasma IL-10 in Plasmodium infected hospital and traditional home patients before treatment compared with the results obtained in the patients after the    treatment (p<0.05; Table1, 2; Figure1).
 * 2) There was a significant decrease in plasma IL-10 in Plasmodium infected hospital and traditional home patients before treatment compared with the control (p<0.05; Table1, 2; Figure1).
 * 3) There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected hospital and traditional home patients before treatment compared with the results obtained in the patients after the    treatment (p<0.05; Table1, 2; Figure1, 2).
 * 4) There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected hospital and traditional home patients before treatment compared with the control (p<0.05; Table1, 2; Figure1, 2).
 * 5) There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected patients in  traditional homes compared with the results obtained  Plasmodium infected  hospital patients(p<0.05; Table1, 2; Figure1, 2).
 * 6) There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected traditional home patients after treatment compared with the non-Plasmodium infected Control(p<0.05; Table1, 2; Figure1, 2).
 * 7) There was a significant increase in Plasmodium parasite density in Plasmodium infected hospital patients after treatment compared with the non-Plasmodium infected Control (p<0.05; Table1, 2; Figure1, 2).
 * 8) There was no  significant difference  in  plasma  IL-10 in Plasmodium infected  patients before and after treatment in traditional homes compared with the results obtained in hospital patients(p>0.05; Table1, 2; Figure1).
 * 9) There was no  significant difference  in  plasma  TNF-α and  Plasmodium parasite density in Plasmodium infected  traditional patients  before  treatment compared with the results obtained   in the  hospital patients(p>.05; Table1, 2; Figure1, 2).
 * 10) There was no  significant difference in plasma IL-10 and TNF-α  in Plasmodium infected hospital  patients after treatment compared with the non-Plasmodium infected Control(p>0.05; Table1, 2; Figure1).
 * 11) There was no significant difference in plasma IL-10 in Plasmodium infected traditional home patients after treatment compared with the non-Plasmodium infected Control (p>0.05; Table1, 2; Figure1).

Figure1: Comparative description of the plasma values of IL-10 and TNF-α, in the subjects

Figure2: Comparative description of the plasma values of Plasmodium parasite density in the subjects

Discussion
There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected hospital and traditional home patients before treatment compared with the control. There was a significant decrease in plasma IL-10 in Plasmodium infected hospital and traditional home patients before treatment compared with the control.

Plasmodium infection is associated with inflammatory response leading to increase in pro-inflammatory cytokines like TNF-α and a decrease in anti-inflammatory cytokines like IL-10 as Tumor necrosis factor alpha (TNF-α) regulates immune cells released by the microphage upon infection to induce inflammatory response to stimulate phagocytosis.[23][24][25][26][30] while Interleukin 10 (IL-10) is an anti-inflammatory cytokine which enhances antibody production[27][28][30] and inhibits the production of pro-inflammatory cytokines such as TNF-α [27][28][30].

There was a significant decrease in plasma IL-10 in Plasmodium infected hospital and traditional home patients before treatment compared with the results obtained in the patients after the treatment. There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected hospital and traditional home patients before treatment compared with the results obtained in the patients after the treatment. These findings can be attributed to the fact that the leaves of Morinda lucida contains phytochemicals with anti-plasmodia, antioxidant and anti-inflammatory bioactivities [1][2][3][4][5][6].

There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected patients in traditional homes compared with the results obtained Plasmodium infected hospital patients. Differences in socio-economic background including treatment seeking behaviour may account for this as reported by Custodio et al.,[31] and not consistent with the report of Rachel et al.,[32] that there was no evidence for an association between infection prevalence and socio-economic status.

There was a significant increase in plasma TNF-α and Plasmodium parasite density in Plasmodium infected traditional home patients after treatment compared with the non-Plasmodium infected Control. There was a significant increase in Plasmodium parasite density in Plasmodium infected hospital patients after treatment compared with the non-Plasmodium infected Control. These findings might be associated with the fact that the parasite has not been totally cleared off the body after treatment. These differences might account for parasite clearance because of the pharmacokinetic-pharmacodynamics modelling of anti-malarial drug effects on parasite clearance predicting therapeutic responses and dose-optimization as explained by Nicholas [33].

A significant reduction in parasite density, TNFα and a significant increase in IL-10 in this work is consistent with the report of Adewole et al., [34]   that  affirms that  M. lucida  has pharmacological activities which include  antimalarial, antidiabetic, hypotensive, anti-inflammatory, immunostimulatory, antioxidant, antimicrobial, antiproliferative, cognitive-enhancement, anti-sickling, anti-trypanosomal, anti-onchocercal, muscle relaxant, antifungal and anti-leishmanial activities.

A significant reduction in plasma TNFα and a significant increase in IL-10 agree with the report of Frederick et al.,  [35]  that reported that in rats M. lucida   showed very good activity against localized and systemic inflammation. reduced interleukin (IL)-1β and tumor necrotic factor, but elevated levels of anti-inflammatory cytokine IL-10 in vitro.

Acknowledgements
Patients of the herbal homes used and Baptist Medical Centre Saki, Nigeria

Competing interests
No competing interest

Ethics statement
The proposal was reviewed and approved by the Research and Ethics Committee of Baptist Medical Centre, Saki, Nigeria before the commencement of the work.(BMC/IX/102) The consent of each subject was also obtained.