In Vivo Anti-Anemic Effect of an Aqueous Root Extract of Phyllanthus Muellerianus (Kuntze) Exell in Model Rats.

Ethnopharmacological relevance Anemia is a very serious condition in Zambia. One of the plants that has been used traditionally is Phyllanthus muellerianus where different parts of shrub are used to treat a number of diseases in Zambian folklore medicine. Earlier studies have investigated medicinal properties of its aqueous root extracts. This study evaluated the effect of P. muellerianus roots on the hematological indices of albino rats and determined its phytochemical prole. medicine in the management of anemia.


Introduction
Anemia is a condition in which the number of red blood cells (and consequently their oxygen-carrying capacity) is insu cient to meet the body's physiologic needs [1][2][3] . The cut off points for diagnosis of anemia have remained largely unchanged since 1968 with the exception that the original age group of children 5-14 years of age was split, and a cut-off of 0.5 g/dL lower was applied to children 5-11 years of age to re ect ndings among non-iron de cient children in the USA 4 . The cut offs range from children 6-59 months (10-10.9 g/dL), children 5-11 years (11-11.4 g/dL), children 12-14 years (11-11.9 g/dL), non-pregnant women 15 years and above (11-11.9 g/dL), pregnant women (10-10.9 g/dL) and men 15 years and above (11-12.9 g/dL) 1,5 It is a common blood disorder that affects people of all ethnicity and ages; although the elderly, young women of child bearing age and infants at greater risk 6 .The causes of anemia are patho-physiologically diverse and multifactorial. Thus, there are more than 400 types, some are mild while others are severe or even life threatening if not treated 7 . In rodents, symptoms include; rapid or labored respiration, anorexia, immobility, abnormal appearance or posture periocular and nasal porphyrin discharge 8 . Treatment varies depending on the type of Anemia. Anemia associated with a serious disease is treated by treating the underlying disorder. Additional medications that boost RBCs may be prescribed when symptoms persist or worsen. These include; iron tablets, supplements, forti cations, erythropoietin injections, blood transfusions, removal of the spleen, plant products such as groundnuts, tomatoes and spinach, animal products such as liver and red meat 5,9,10 etc. Interventions to prevent or treat anemia are insu cient in Zambia because of; inadequate quali ed human resource, high disease burden, inadequate emergency facilities, the diet of majority Zambians is mainly composed of cereals (maize) and starchy roots with little micronutrient-dense foods such as animal products and fruits 11,12 . Further, the prevalence of anemia is 46 % which is a severe public health problem based on the World Health Organization (WHO) standards. This implies that there is a great loss of man hours of healthy adults who nd themselves off work to nurse anemic patients. It is also among the top 10 causes of morbidity and mortality 3,[13][14][15][16] . In Africa and most Asian countries, anemia is treated using herbs such as; -Khaya senegalensis, Justicia secunda, and Amaranthus spinosus 17 .
The study investigated the effect of Phyllanthus muellerianus (Kuntze) Excell aqueous root extract on hematological indices of albino rats. P. muellerianus is one of the plants used to treat anemia by local people of the Northern part of Zambia. However, its e cacy had not been scienti cally established. Local names for P. muellerianus in Zambia include: Chewa-Mkuzandola, Tumbuka-Kapikanduzi 18 , Icibemba-Umupetwalupe, Kaonde-Mulembalemba, Mambwe-Mupetwandupe. It belongs to the family Phyllanthaceae consisting of approximately 1000 species which are widely distributed in tropical and subtropical areas of Africa, Asia, America and Australia. It is an evergreen scandent shrub with numerous stems from the base or a small tree up to 12 meters tall. The branches are arched and pendulous almost to the ground. It naturally occurs in riverine forest and wooded grasslands on deep and well-drained soils.
It is widely distributed and easy to access in Zambia. Additionally P. muellerianus grows easily from seed and hence has potential to contribute sustainably towards medical solutions coming from local ora 19 .
P. muellerianus has many medicinal uses such as the treatment of wounds, menstrual disorders, fevers, in ammation, intestinal problems, kidney and urinary bladder problems, diabetes and hepatitis B, body pain and as an antiseptic 20 .
Agyare et al 21 studied P. muellerianus leaf extracts for the stimulatory effect of ellagitannins on cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal broblasts. Earlier studies on the leaf extract by Boakye et al 22 have shown that P. muellerianus has anti-in ammatory activity. The review by Calixto and friends 23 showed that the phyllanthus species have a number of metabolites with pharmacological potential isolated and characterised from all the parts of the plant, leaves, roots, stem and bark. Other studies investigated the antimicrobial properties of the stem and bark parts of 24,25 . This study investigates the ethnobotany and ethnopharmacological effect of P. muellerianus roots on hematological indices of albino rats.

Plant Collection and Identi cation
Ethnobotanical authentication and annotation was done at the University of Zambia Herbarium (UZL). The specimen was identi ed as Phyllanthus muellerianus (Kuntze) Exell belonging to the family Phyllanthaceae and voucher specimen/accession number 22287. It has also be veri ed on the http://www.theplantlist.org website.

Preparation of the aqueous root extract of the plant
The roots of the plant were harvested in October 2018 from Kaunda Square area of Lusaka Province (Latitude 15°21'33.1"S and Longitude 28°21'57.9"E), Zambia. They were thoroughly washed to remove debris and all soil material. After size reduction, a 1 kg root sample was boiled in 1.5 litres of distilled water and the resulting solution was allowed to cool then sieved to remove non-soluble plant matter and nally ltered through Whatman lter paper number 4. The mixture was boiled to dryness on a heating mantle, the resulting brittle powder was weighed and kept at 4 o C until further use. Various concentrations (w/v) of the extract were made by dissolving the appropriate quantity of the solid extract in distilled water to make a solution.

Animals and induction of Anemia
Sample size was calculated by executing the function in G*Power version 3.1.9.4 26, 27 . Brie y, under test family, "F test" was selected, and the statistical test selected was "ANOVA: Fixed effects, omnibus, oneway". Sample size was then computed as a function of power level (1 -β), a pre-speci ed signi cance level (α) and the population effect size to be detected with probability. The power level (1 -β) was set at 0.8 default, signi cance level α at 0.05 default and pre-detected effect size at 0.7, and 6 groups.
Calculation using G*Power gave a sample size of 36. So a total of 36 male albino experimental animals weighing between 150 and 180 grams were selected and put into 6 treatment groups with 6 animals each using similar studies by 28 ..All laboratory work was done according to the Guidelines for the Care and Use of Laboratory Animals 29 . Anemia was induced by bleeding the rats under light anesthesia using diethyl ether as an anesthetic 30, 31 . The formula described by Lee and Blaufox 32 was used to determine the quantity of blood removed through bleeding. At the end of the study, rats were euthanized in diethyl ether 33 .

Administration of the plant extract and Hematological tests
Treatment started 24 hours after inducing anemia. Ranferon, the extract and distilled water were administered by oral intubation as shown in Table 4 − 1. Rats were anesthetised in diethyl ether, when they became unconscious, blood was collected from their retro-orbital plexus for hematological studies 34 at the baseline of the study, after inducing anemia and after treatment 35, 36 .

Phytochemical Screening
Qualitative phytochemical screening of the aqueous plant extract was carried out at the University of Zambia, Department of Chemistry using standard procedures 37, 38 .

Mineral quanti cation and Statistical analysis
The mineral content was determined by AAS using Perkin-Elmer atomic analyst 400 while oneway ANOVA followed by Bonferroni post hoc test to determine pairwise comparisons was executed in STATA software version 13.0. In each case, the negative control was compared with other categories. All results were expressed as percent means at 95% con dence level with respective standard deviations.

Quality control
To ensure reliability of results, quality control was performed on the AAS and hematocrit before each test was done according to the respective manufactures manual.

Results
The color of solid extract was dark brown and the percentage yield was 1.6 %. The extractive weight of the plant extract was 16 g with a mineral composition of 230.5 mg Fe, 273.5 mg Mn, and 138 mg Zn.

Phytochemical Screening
The results of phytochemical screening of the extract are shown in

Effects of the plant extract on the Packed Cell Volume and Hemoglobin
The study de ned anemia as Hb < 12 g/dL. At the baseline of the study, the mean values for PCV and Hb in the 5 comparison groups were not different (P > 0.05) from the control group. However, repeated bleeding decreased PCV and Hb to values less than 32.5 % and 11.5 g/dL respectively. Treatment with P. muellerianus reversed anemia in a dose-dependent manner ( Fig. 4 − 1 to all other groups. There were also differences when the normal control was compared to 400 mg/kg. Ranferon was lower compared to 400mg/kg, 100mg/kg was lower compared to 200 mg/kg and 400 mg/kg. Also the 200mg/kg was lower when compared to the 400 mg/kg extract (Fig. 4 − 2).

Effects of the plant extract on MCV, MCH and MCHC
The mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and the mean corpuscular hemoglobin concentration (MCHC) all showed no statistical differences across the groups at 95 % con dence level.

Effect of P. muellerianus on the hematological parameters
Dosages of 100, 200, and 400 mg/kg of the root extract and ranferon were administered orally to anemic albino rats to monitor their effect compared to that of the control group that did not receive any drug but distilled water. The results revealed that the root extract and ranferon were able to restore the hematological indices of experimental animals to normal levels and the signi cant (p < 0.05) effect was found to be dose-dependent. In addition, the eta squared values for PCV and Hb of 0.66 suggested that the aqueous root extract of the plant was both statistically signi cant and e cacious against anemia. The Phytochemical and mineral compositions of the root extract seem likely to be responsible for the hematinic effect of P. muellerianus and their presence in the plant extract agrees with previous studies 39,40 .The blood parameters, Hb, PCV, and RBCs together with the level of iron are indices of anemia that could be used to indicate nutritional values of ingested diets as well. MCV, MCH and MCHC are constants for typing anemia hence they were not statistically different (p > 0.05) when experimental groups were compared to the control group after treatment. They, however, decreased after inducing anemia, indicating microcytosis as their decrease re ects a release of RBCs, which are less saturated in Hb (hypochromia). Therefore, occurrence of anemia observed in this study was attributable to lowered values of these indices and related to the report by Osman et al., 41 .Moreover, the increase in these hematological parameters was dose-dependent and could be due to high nutritional values of P. muellerianus particularly in minerals such as Fe (230.5 mg). Fe plays a signi cant role in erythropoiesis. It is required for the synthesis of Hb and myoglobin while its de ciency causes anemia. However, the therapeutic potential of P. muellerianus could not be established based on available Fe content alone as other factors play a role in its absorption in the body. In this context, Fe is a necessity for the formation of  42 to have a lethal dose (LD 50 ) of more than 4 g/kg body weight of male and female rats. Based on their study, they concluded that P. muellerianus is not toxic, taking into consideration the 5 g/kg threshold of toxic substances. On the other hand, Adedapo et al., 43 showed that the leaves of P. muellerianus signi cantly (p < 0.05) reduced the hematological parameters, had toxic potential and were therefore, poisonous to the animals.

Observed Phytochemicals and their action
Plants used in the treatment of disease contain a wide range of active principles with biological activity, some of which are responsible for the characteristic odours, pungencies and colours of plants while others give a particular plant its culinary, medicinal or poisonous virtues, which could be used as the base for discovering modern drugs for curing various diseases 39 . The phytochemical tests performed on the root extract of P. muellerianus showed presence of steroids, triterpenoids, alkaloids, avonoids, saponins, cardiac glycosides and tannins as reported by 20,39,44 in previous similar studies.

Sterols
Phytosterols (PS) such as β-sitosterol, campesterol and stigmasterol 45 have been shown to have anti eryptotic effects on cells. Presence of a considerable amount of sterols may have had the anti hemolytic property hence improving availability of intact cells in blood, ultimately alleviating anemia.

Alkaloids
As inferred from other reports, alkaloids, the most revered of all phytochemicals are said to be pharmacologically active and their action is felt in blood vessels. They inhibit cyclic adenosine monophosphate (cAMP) phosphodiesterase leading to accumulation of cAMP. This effect stimulates phosphorylation of proteins and synthesis of proteins, which improves erythropoiesis 39, 40 .

Saponins
Saponins are known to vitalize blood circulation and promote hemolytic activities. Since saponins are active agents that lyse the membrane of RBCs, it is likely that the plant extract used in this study rst lysed RBCs. Then the test animals overcame this inhibition by producing a glycosidic enzyme that cleaves some of the terminal sugars from the saponins, which detoxi ed it 39 . This detoxi cation of saponins reinforced the proper use of iron contained in the plant extract allowing it to synthesize hemoglobin for new RBCs, thus leading to an observed improvement of Hb, RBCs and PCV in the plant extract treated groups. Glycosides enhance the natural resistance and have the recovery powers for the body 39 .

Flavonoids
Flavonoids are known to possess a well-established protective effect against membrane lipoperoxidative damages. The antioxidant activity of phenols and avonoids is mainly attributed to their redox properties because of which they act as reducing agents, electron / hydrogen donators, and singlet oxygen quenchers. It has been demonstrated that the antioxidants such as avonoids can act: either by neutralizing reactive oxygen species (ROS) by directly reacting with superoxide anion, nitric oxide and peroxynitrite thereby preserving vascular function and protecting vascular injuries from ROS and perhaps from other oxidant species, or they could stimulate erythropoiesis 46 .

Tannins
Tannins are well known for their anti-oxidant and skin regeneration.

Consent for publication
All the authors consented to publication of this manuscript.

Availability of data and material
Not applicable as all data is presented herein.

Competing interests
The authors declare no competing interests.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.
Author contribution GML, collected samples and performed experiments and contributed to writing the manuscript, SG contributed to writing the manuscript, DKC collected, accessioned the plant sample in the UZL and contributed to writing the manuscript, JN and KMM designed experiments and critically analyzed data. JN contributed to overall supervision. All authors read the manuscript and contributed to editing.

Figure 1
Mean Packed Cell Volume after treatment with the extract. The asterisks imply signi cance across the treatment groups. The results were expressed as mean ± SD at 95% con dence level, p<0.05 (Oneway ANOVA followed by Bonferroni post hoc test executed in STATA version 13.1) The plot was generated using GraphPad Prism software version 6.01.

Figure 2
Shows mean Hb levels The asterisks imply signi cance across the treatment groups. The results were expressed as mean ± SD at 95% con dence level, p<0.05(Oneway ANOVA followed by Bonferroni post hoc test executed in STATA version 13.1) The plot was generated using GraphPad Prism software version 6.01. Figure 3 shows the results of treatment of the rats with extract. The Bonferroni post hoc test revealed that signi cance was only between the 100 and 400 mg/kg treatment groups. Double asterisks shows signi cance between these two groups. The results were expressed as mean ± SD at 95% con dence level, p<0.05 (Oneway ANOVA followed by Bonferroni post hoc test executed in STATA version 13.1). The plot was generated using GraphPad Prism software version 6.01.