Bioactive Natural Constituents from Lemongrass Tea and Erythropoiesis Boosting Effects: Potential Use in Prevention and Treatment of Anemia

Abstract

This study assessed the effects of lemongrass (Cymbopogon citratus) tea on hematologic indices in human volunteers. One hundred five subjects (55 men and 50 women), aged 18 to 35 years, were randomly assigned to groups set to orally receive infusion prepared from 2, 4, or 8 g of C. citratus leaves once daily for 30 days. Assessment of hematologic indices (hemoglobin concentration [Hb], packed cell volume [PCV], red blood cell [RBC] count, mean cell Hb [MCH], mean cell volume [MCV], mean cell Hb concentration [MCHC], total white blood cell [WBC-total] and differentials, and platelets) were performed 1 day before (baseline), and at 10 (acute) and 30 days (subchronic phase) after the initiation of treatment. Results obtained on days 10 and 30 were compared with baseline values. Infusions prepared from C. citratus leaf powder, which tested positive for tannins, saponins, alkaloids, flavonoids, macro- and micronutrients, significantly increased PCV, Hb, and RBC (P<.05) in all subjects, particularly in the subchronic phase of the study. MCH, MCV, and MCHC were not significantly different from baseline values in both the sexes. WBCs and differentials significantly decreased (P<.05) with the exception of neutrophils and lymphocytes, which significantly increased in some or all groups (P<.05), respectively. C. citratus leaf infusion appears to exert an erythropoiesis boosting effect, likely due to some nutritional constituents and its antioxidant and pharmacologic properties.

Introduction

The use of bioactive natural constituents from food sources as a new strategy for prevention and management of hematologic disorders associated with qualitative and quantitative deficiency of hemoglobin (Hb) and red blood cells (RBCs) has gained a global significance.¹ This is not only because of the significant drawbacks that characterized the use of synthetic hematinics but also because of the increasing awareness and interest in the health benefits derived from herbs and natural products.^2,3

Medicinal plants are rich sources of nutrients (macro and micro) and natural bioactive substances with vast potentials for improving humans' health and preventing acute and chronic diseases through their immunity boosting, antioxidant, anti-inflammatory, antimicrobial, anticarcinogenic, antisickling, antihelminthic, antimalarial, antipyretic, analgesic, and erythropoiesis boosting effects.⁴ An extract containing some or all these properties may provide therapy for secondary causes of anemia while the nutrient constituents may serve as precursors for hematopoiesis. One such plant is Cymbopogon citratus, an economically important, essential oil-yielding, aromatic perennial plant of the Poaceae family that is widely distributed and consumed globally. Originally native to Southeast Asia, C. citratus is now found in many continents and countries, including South America, Africa, India, and Australia.⁵ Nutritionally, C. citratus contains variable amounts of electrolytes and minerals (sodium [Na²⁺], potassium [K⁺], calcium [Ca²⁺], copper [Cu²⁺], magnesium [Mg²⁺], manganese [Mn], selenium [Se], phosphorus [P], iron [Fe²⁺], and zinc [Zn²⁺]), vitamins (folate, niacin, pyridoxine, riboflavin, and vitamins A, C, and E), and macronutrients (carbohydrate, protein, and small amounts of fat).^5,6 These rich nutrient constituents support its use in aromatic drinks and traditional cuisine, including as tea, nonalcoholic beverages, ice cream, candies, pastries, desserts, chewing gums, and other baked foods and confections.^7,8 Its pharmacologic actions include antibacterial,⁹ antifungal,¹⁰ antiprotozoal,¹¹ anticarcinogenic,¹² anti-inflammatory,¹³ antioxidant,¹⁴ antidiabetic,¹⁵ antihelminthic,¹⁶ antimalarial,¹⁷ anti-influenza, antipyretic, and antipneumonia effects.¹⁸ These actions are mediated by its phytochemical and essential oil constituents, tannins, saponins, flavonoids, and citral, and so on. Citral is the most abundant (65%–85%) essential oil constituent and appears to be the most pharmacological and physiological importance. It is a mixture of two geometric isomers, geraniol and neral, and is responsible for the aromatic scent of the plant.^19,20 The aforestated pharmacologic actions of C. citratus can exert therapeutic effects on the secondary causes of qualitative and quantitative deficiency of Hb and RBCs.^16,17 Thus, C. citratus through its phytochemical constituents can eliminate the secondary causes and prevent anemia while the nutritive constituents may serve as precursors for hematopoiesis.

In some developing countries, high prevalence of anemia is associated with acute and chronic diseases and poverty leading to poor nutrition and inaccessibility to synthetic drugs. This plant may serve as the primary source of nutrients and provide therapy for the secondary causes of anemia. Regrettably to date, no study has investigated the effects of C. citratus on hematologic indices in humans, despite its widespread nutritional and therapeutic uses in traditional medicine. Additional evidence is needed to make a strong case for its use. Here, we tested the effects of infusions prepared from C. citratus leaves on hematologic indices in human volunteers. We also evaluated its phytochemical/nutritional constituents, all of which are known to influence hematopoiesis.

Materials and Methods

Plant materials: collection, identification, and preparation of C. citratus leaf extract

Fresh C. citratus leaves were obtained from an agricultural farm in Uyo, Akwa Ibom State, Nigeria, few days before utilization, in the month of May 2012. The leaves were identified and authenticated by a taxonomist in the Department of Botany at the University of Uyo. Voucher specimen No. UUH3276/UYO was deposited at the herbarium in the Department of Botany at the University. The leaves were rinsed, sun-dried, and pulverized into powder using electric blender to give a gram weight of 200 g. This was soaked in a container with 2 L of hot water and allowed to stand for about 8 h. Thereafter, the solution was filtered using No. 2 Whatman filter paper. The filtrate was evaporated by heating in water bath at 40°C to obtain the solid extract. The solid extract was weighed with an electric weighing balance (ACS-ZE14; Surgifriend Medicals Ltd., England, UK) to obtain a yield of 70 g (35% w/w), which was stored in clean bottles at room temperature until required for use. Similar procedures were repeated using 2, 4, and 8 g of C. citratus powder and yields of 410, 810, and 1570 mg extract were obtained, respectively.

Phytochemical screening of C. citratus leaf extract

The phytochemical analysis of the extract was carried out using the standard procedures to determine the levels of saponins, phenolics, alkaloids, tannins, flavonoids, glycosides, steroids, deoxy sugars, and anthraquinones.^21,22

Determination of nutrient constituents

Ash, crude fiber, fat, moisture, and protein contents were determined by proximate analysis using procedures provided by the Association of Official Analytical Chemists.²³ To determine the levels of mineral constituents, extract aliquots were predigested with concentrated HNO₃, followed by digestion with a mixture (10:3) of concentrated HNO₃ and concentrated HClO₄. The acid samples were heated in a covered 50-mL beaker until all traces of HClO₄ were eliminated, as reflected by the absence of white fumes. The final liquid in the digestion beaker (about 2 mL) was brought up to a volume of 25 mL with deionized water and assayed for copper, iron, magnesium, sodium, and calcium by using an atomic absorption spectrophotometer (Jarrel-Arh model 82-362). Determination of water-soluble vitamins in the extract was performed according to the methods described by Ismail and Fun.²⁴ A reverse-phase high-performance liquid chromatography (RP-HPLC) (model 582 ESA, Inc., Chelmsford, MA, USA) was used to identify the water-soluble vitamins using an aqueous mobile phase method.²⁵

Determination of antinutrient constituents

Levels of the following antinutrients were also assessed using 100 g of C. citratus leaf extract: tannins, hydrogen cyanide, phylate, and oxalate. The presence of tannic acid (a polyphenol) was determined using the method described by Makkar and Goodchild.²⁶ Hydrogen cyanide was determined using the method described by Fasuyi and Nonyerem.²⁷ The presence of phylate was determined using the method described by Young and Greaves.²⁸ The presence of oxalate was determined by the titrimetric method of Moir.²⁹

Study design

This study used a pre- and postexperimental design to assess the effect of C. citratus leaf infusion on the hematologic indices of healthy adult humans (55 men and 50 women) who were selected by a simple random technique. The informed written consent was obtained from all participants. All participants underwent a thorough presurvey medical screening performed by a physician to ensure medical fitness and to exclude those who did not meet the inclusion criteria. The exclusion criteria are as follows: inappropriate age, a history of kidney or liver disease, failure to satisfy the preresearch clinical and biochemical assessment, pregnancy or lactation, allergy to any lemongrass constituents, and use of drugs known to affect or to be metabolized primarily in the kidney. Screening included determination of medical history, lifestyle assessment (such as smoking, drinking, physical activity, diet, and drug history), BP and heart rate (HR), weight, blood glucose level, full blood and platelet count, and urine and blood indices of renal and hepatic function. The participants were advised to avoid excessive physical activity and ingestion of drugs or alcohol and to remain on their regular diet throughout the study period. The study protocol was approved by the Institutional Research Ethics Committee, and the study was conducted in the Department of Physiology at the University of Uyo, Nigeria, according to the rules set forth in the Declaration of Helsinki governing the conduct of human research.

Safety evaluation/dose determination and administration of infusion

The participants were subdivided into three groups (n=35/group). Groups 1, 2, and 3 received infusions prepared from 2, 4, or 8 g of C. citratus leaf powder in 150 mL of hot water, respectively, given once daily for 30 days. The infusion was prepared as usual employed by the population to prepare lemongrass spices, ethnomedicine, and recreational teas, soups, and curries.³⁰ The dose range was adapted from previous human studies^31,32 in which participants exhibited no obvious clinical or biochemical evidence of toxicity. To further confirm the safety of this dose range, the investigators of the present study conducted a pilot survey on 10 human volunteers using 2, 4, 8, and 10 g of C. citratus leaf powder. Participants who had received at least a dose of the infusion prepared from 2, 4, or 8 g of C. citratus in equal volume of hot water (150 mL) daily for 1 week showed no evidence of adverse/toxic effect, as judged by the results of the tolerability evaluation.

Tolerability evaluation consisted of a range of clinical and laboratory tests, including tests for liver function (aminotransferase activity), renal function (serum creatinine and clearance rate [Cr]), serum urea levels, and hematologic indices. Physical examinations were also performed on each participant to check for the presence of jaundice or pallor (evidence of hepatotoxicity or hemolysis) as well as abnormal skin reaction. Adverse events reported by the participants or observed by the investigators during the clinical evaluation were recorded. Also, participants were closely monitored during the study period. They were submitted a daily symptomatology questionnaire, which contained 25 questions designed by the authors. The presence of symptoms, such as lightness of the body, blurring of vision, insomnia, headache, dizziness, sweating, frequent micturition, belching, dyspepsia, diarrhea, constipation, vomiting, increase thirst, and awareness of fast heart beating (palpitation), were to be reported.

The authors had chosen 2 g of C. citratus leaf powder as the starting dose to correspond to the quantity usually employed by the Brazilian population to prepare their lemon grass tea.³¹ Studies by others have shown that in Brazil, lemon grass tea or infusion, prepared by pouring 150 mL of boiling water over 2–3 g fresh or dried leaf of lemon grass, is one of the most popular remedies in their traditional medical practices for treating “nervous disturbances such as insomnia, irritability, and anxiety.” To evaluate the dose effect, the 2 g was doubled and quadrupled to give 2, 4, or 8 g of C. citratus leaf powder used in the present study.

Schedule of data collection

Venous blood samples were obtained for hematologic and biochemical analysis. The hematologic parameters measured included packed cell volume (PCV), Hb, RBC, mean cell Hb (MCH), mean cell volume (MCV), mean cell Hb concentration (MCHC), white blood cell (WBC)-total and differentials, and platelets. The measurements were performed within 2 h of sample collection using the SYSMEX KX-21 Automated Hematology Analyzer (Kobe, Japan) in the Hematology Unit at the University of Uyo Teaching Hospital.

Biochemical parameters measured were serum creatinine (SCr), urea and uric acid, liver enzymes (aspartate aminotransaminase, alanine transaminase, and alkaline phosphatase), bilirubin, protein, glucose, and serum lipid profile (HDL-C, LDL-C, VLDL-C, TG, and Chol). Serum creatinine level was determined by the Jaffe's method using 0.75 NaOH and 1% Picric acid (Sigma Chemicals, Thane, India) at a volume of 1 mL each to the serum-specific specimen. A standard was similarly treated. A color change that developed within 15 minutes at room temperature was measured spectrophotometrically (ESA, Inc.) at 520 nm. Serum total cholesterol (T-chol), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), and glucose were measured using the lipid profile and glucose automated measuring system (Lipid Pro™), Model ILM-0001 A; Infopia Co. Ltd., Hogye-dong, South Korea). Serum uric acid and urea were measured using the multichannel automated analyzer (SYNCHRON, Los Angeles, CA, USA). Renal function (estimated glomerular filtration rate [eGFR]) was assessed primarily by using serum creatinine, estimation of glomerular filtration, and estimation of creatinine clearance calculation using the modified Cockcroft and Gault formula. All measurements were performed 1 day before and at 10 and 30 days after the start of infusion administration.

Statistical analysis

The statistical analysis of data was performed using one-way analysis of variance statistical technique (one-way ANOVA). Furthermore, for the purpose of pairwise comparison, the least significant difference (LSD) test was performed and a probability value less than 5% (P<.05) indicates statistical significance. All statistical computations were enhanced using the Statistical Package for Social Sciences (SPSS 20.0). Results obtained are presented as mean±standard error of the mean (SEM).

Results

Phytochemical constituents of C. citratus leaf extract

The results of the phytochemical analysis of the hot water extract of C. citratus show that it contains relatively high concentrations of saponins; moderate levels of tannins, flavonoids, and phenols; and relatively low concentrations of anthraquinones, alkaloids, and deoxy sugars (Table 1).

Table 1.

Phytochemical Constituents of Cymbopogon citratus Leaf Extract

Phytochemical constituents	Concentration
Saponins	+++
Tannins	++
Flavonoids	++
Phenols	++
Anthraquinones	+
Alkaloids	+
Deoxy sugars	+
Steroids	−
Cyanate	−
Phlobatannins	−

−, absent; +, present; ++, moderate; +++, marked.

Analysis of nutrient constituents of C. citratus leaf (per 100 g)

Table 2 shows the nutritional constituents of C. citratus leaf (per 100 g), including moisture, crude protein, fiber, fat, carbohydrate, and micronutrients, of which K⁺, Ca²⁺, and Mg²⁺ were present in higher concentrations. Vitamins (C and B group) were also present.

Table 2.

Analysis of Nutrient Constituents of C. citratus Leaf (Per 100 g)

Nutritional constituents	Nutritional value
Constituents
Moisture	2.36 g
Total ash	4.20 g
Crude protein	2.65 g
Crude fiber	3.04 g
Carbohydrates	36.02 g
Total fat	0.65 g
Cholesterol	0 mg
Electrolytes
K⁺	475 mg
Na⁺	8 mg
Minerals
Ca²⁺	56 mg
Fe²⁺	15.6 mg
Mg²⁺	46.20 mg
Cu	0.386 mg
Mn	8.34 mg
Se	1.02 mcg
Zn²⁺	2.05 mg
Vitamins
Vitamin C	3.8 mg
Folate	88 mcg
Thiamine	0.84 mg
Niacin	2.01 mg
Pyridoxine	0.04 mg
Riboflavin	0.186 mg

Baseline demographic characteristics of the study participants

Table 3 shows the baseline demographic and clinical characteristics of the study participants. Most of them (52%) were males, between the ages of 18 and 35 years, from Ibibio ethnicity and Christians. Their mean weight, BMI, SBP, DBP, MAP, and HR were 60.74±1.93 kg, 23.4±0.75 kg/m², 120.53±1.89 mm Hg, 74.64±1.6 mm Hg, 85.69±1.13 mm Hg, and 77.71±1.99 beats/min, respectively, whereas their mean respiratory rate and eGFR were 18.56±1.52/min and 99.88±1.52 mL/min, respectively.

Table 3.

Baseline Demographic Characteristics of the Study Participants

Characteristics	Baseline values
Demographic characteristics
Average age (SD)	27.42±0.30
Age range (years)	18–35
Sex
Male	55 (52.0)
Female	50 (48.0)
Race (% black)	105 (100)
Ethnic (% Ibibio)	105 (100)
Religion (% Christianity)	105 (100)
Clinical characteristics
Weight (kg)	60.74±1.93
Height (m²)	1.62±0.01
BMI (kg/m²)	23.46±0.75
SBP (mm Hg)	120.53±1.89
DBP (mm Hg)	74.64±1.62
MAP (mm Hg)	85.69±1.13
Heart rate (beats/min)	77.71±1.99
Pulse pressure (mmHg)	45.89±1.04
Respiratory rate (/min)	18.56±1.52
eGFR (mL/min)	99.88±1.52

Values are expressed in mean±SEM.

Effects on plasma lipid profile and serum liver enzymes of the study participants

The serum biochemical profile of the study participants are shown in Tables 4 and 5.

Table 4.

Acute (10 Days) and Subchronic (30 Days) Effects of C. citratus Leaf Infusion on Fasting Blood Glucose and Serum Lipid Profile of the Study Participants

		Serum lipid profile
Parameters	Blood glucose (mM)	Chol (mM)	TG (mM)	HDL (mM)	LDL (mM)
Control
2 g	5.18±0.14	4.20±0.01	0.88±0.02	0.82±0.01	1.80±0.07
4 g	5.31±0.13	3.80±0.03	0.96±0.01	0.98±0.04	2.01±0.09
8 g	5.61±0.21	4.00±0.01	0.82±0.05	0.94±0.02	2.24±0.05
Acute phase
2 g	5.40±0.09	4.04±0.06^a	0.82±0.03	0.90±.02^a	1.08±0.04
4 g	5.30±0.14	3.04±0.04^{a b}	0.84±0.01^a	1.02±0.06	1.98±0.08
8 g	5.23±0.09	3.02±0.01^{a b}	0.80±0.01^c	1.01±0.09^a	2.06±0.02
Subchronic phase
2 g	5.05±0.10	3.98±0.03^a	0.84±0.01	0.90±0.02^a	1.75±0.08
4 g	5.13±0.15	3.20±0.05^{a b}	0.82±0.01^a	1.02±0.06	1.88±0.12^{a b}
8 g	5.22±0.24	3.34±0.02^{a b c}	0.76±0.04	1.10±0.04^b	1.98±0.08^{a b}

Values are expressed in mean±SEM.

Significantly different from control (P<.05).

Significantly different from 2 g.

Significant different from 4 g.

TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Table 5.

Acute and Subchronic Effects of C. citratus Leaf Infusion on Serum Liver Enzymes, Bilirubin, Total Serum Protein and Uric Acid Levels of the Study Participants

	Liver enzymes
Parameters	AST (μ/L)	ALP (μ/L)	ALT (μ/L)	Bilirubin (μM)	Protein (g/L)	Uric acid (mM)
Control
2 g	12.94±0.58	23.34±0.63	7.40±0.42	9.59±0.68	67.09±0.53	0.40±0.02
4 g	12.00±0.42	22.54±0.58	7.83±0.47	12.55±0.93	67.11±0.62	0.30±0.01
8 g	12.54±0.60	23.23±0.42	7.57±0.59	10.17±2.71	67.77±0.88	0.41±0.02
Acute phase
2 g	11.17±0.87	24.34±0.59	8.06±0.33	12.57±0.95^a	68.69±0.66	0.39±0.02
4 g	13.20±1.79	24.71±0.61	8.74±0.59	15.83±1.32^{a b}	69.23±0.75	0.37±0.0^a
8 g	10.63±1.09	23.83±0.76	8.31±0.42	19.32±2.17^{a b}	68.71±0.68	0.39±0.02
Subchronic phase
2 g	10.83±0.93	31.51±1.95	8.17±0.61	5.89±0.40	67.63±0.72	0.36±0.01^a
4 g	11.00±0.89	30.80±2.13	7.49±0.57	5.33±0.72	68.29±0.60	0.28±0.02^a
8 g	7.80±0.95	28.77±1.45	7.49±0.54	4.34±0.24	67.57±0.49	0.27±0.01^a

Values are expressed in mean±SEM.

Significantly different from control (P<.05).

Significantly different from 2 g.

ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate aminotransaminase.

Effects on some hematologic parameters in male and female subjects

Table 6 shows that at day 10, PCV and Hb significantly increased in both male (P=.05) and female (P=.01) subjects treated with an infusion prepared from 8 g of C. citratus leaf powder. At day 30, PCV and Hb significantly increased in male subjects of all groups (P=.05 and 0.01), respectively, whereas a similar increase was found in female subjects who received an infusion prepared from 4 or 8 g of C. citratus leaf powder (P=.01), respectively. At day 10, a significant increase in RBCs was found in male subjects who received an infusion prepared from 4 or 8 g of C. citratus leaf powder (P=.01). Total WBC counts in both male and female subjects of all groups were not significantly different from baseline counts.

Table 6.

Acute and Subchronic Effects of C. citratus Leaf Infusion on Some Hematologic Parameters of the Study Subjects

	Male				Female
Parameters	PCV (%)	Hb (mg/dL)	RBC (mm³)	WBC (mm³)	PCV (%)	Hb (mg/dL)	RBC (mm³)	WBC (mm³)
Baseline (control)
2 g	38.71±0.92	12.90±0.21	5.01±0.12	5.52±0.11	35.74±0.83	11.91±0.23	4.23±0.21	5.01±0.27
4 g	41.02±0.72	13.67±0.27	4.23±0.10	5.89±0.22	38.27±0.75	12.76±0.24	3.25±0.05	5.22±0.23
8 g	38.94±0.65	12.98±0.24	4.02±0.12	5.73±0.16	36.22±0.69	12.07±0.32	3.37±0.09	5.16±0.21
Acute phase
2 g (410 mg yield)	38.62±1.91	12.87±0.43	4.98±0.17	5.43±0.13	34.82±0.74	11.61±0.24	4.22±0.11	4.95±0.17
4 g (810 mg yield)	42.85±0.81	14.28±1.02	5.01±.13^a**	5.84±0.32	39.57±0.63	13.19±0.87	3.17±1.14	5.2±0.19
8 g (1570 mg yield)	42.92±1.78^a*	14.31±0.57^a*	5.72±.17^a**	5.26±0.19	41.96±0.55^a**	13.99±0.25^a**	3.12±1.16	4.97±0.10
Subchronic phase
2 g (410 mg yield)	42.62±0.82^a*	14.21±0.52^a*	5.02±0.21	5.46±0.22	35.01±0.81	11.67±0.27	4.30±0.21	5.01±0.29
4 g (810 mg yield)	48.98±0.95^a**	16.33 0.25^a**	5.02±0.11^a**	5.62±0.41	44.63±0.92^a**	14.88±0.28^a**	4.61±0.05^a*	5.35±0.27
8 g (1570 mg yield)	47.08±0.82^a*b	15.69±0.29^a*b	5.60±0.15^a**	5.58±0.23	46.02±0.44^ab	15.34±0.26^ab	4.75±0.15^a**	5.04±0.31

Values are expressed in mean±SEM.

Significantly different from baseline at 5% (P<.05).

a**

Significantly different from baseline at 1% (P<.01).

a**b*

Significantly different from baseline at 1% (P<.01) and 10 days at 5% (P<.05).

a**b**

Significantly different from baseline and 10 days at 1% (P<.01).

PCV, packed cell volume; RBC, red blood cell; WBC, white blood cell.

Effects on RBC indices (MCV, MCHC, and MCH)

Table 7 shows that MCV, MCHC, and MCH were not significantly different from baseline values in all the groups.

Table 7.

Acute and Subchronic Effects of C. citratus Leaf Infusion on RBC Indices

	Acute (10 days)			Subchronic (30 days)
Concentrations	MCV (mm³)	MCHC (mm³)	MCH (pg)	MCV (mm³)	MCHC (mm³)	MCH (pg)
Baseline (control)
2 g	28.28±0.45	33.20±0.16	28.28±0.45	28.28±0.45	33.20±0.16	28.28±0.45
4 g	26.20±0.33	32.17±0.16	26.20±0.33	26.20±0.33	32.17±0.16	26.20±0.33
8 g	27.03±0.42	33.08±0.18	27.03±0.42	27.03±0.42	33.08±0.18	27.03±0.42
2 g (410 mg yield)	28.67±0.42	33.17±0.19	28.67±0.42	28.96±0.46	33.14±0.23	28.96±0.46
4 g (810 mg yield)	27.57±1.28	32.54±0.30	27.57±2.28	27.37±1.29	32.69±0.24	27.37±0.29
8 g (1570 mg yield)	27.14±0.39	33.00±0.32	27.14±1.39	28.07±0.41	32.60±0.20	28.07±1.41

Values are expressed in mean±SEM.

MCH, mean cell Hb; MCHC, mean cell Hb concentration; MCV, mean cell volume.

Effects on WBC lineages (neutrophil, lymphocyte, basophil, eosinophil, and monocyte counts)

Table 8 shows that there were significant decreases in other WBC lineages, including platelets, compared with baseline values. The exceptions were lymphocytes, which showed a significant increase in groups treated with infusions prepared from 2 or 4 g of C. citratus leaf powder (P=.05), and neutrophils, which increases only in groups treated with infusions prepared from 4 or 8 g of C. citratus leaf powder (P=.05), respectively.

Table 8.

Acute and Subchronic Effects of C. citratus Leaf Infusion on WBC Lineages of the Study Subjects

Parameters	Neutrophil count (%)	Lymphocyte count (%)	Basophil count (%)	Eosinophil count (%)	Monocyte count (%)	Platelet (%)
Baseline (control)
2 g	42.40±1.77	44.91±1.28	0.46±0.09	8.06±0.77	4.54±0.29	183.34±2.02
4 g	40.50±1.42	45.86±1.31	0.46±0.03	8.26±1.14	4.77±± 0.14	157.60±3.24
8 g	37.31±1.97	47.29±1.61	0.40±0.04	10.37±0.27	4.34±0.45	178.63±6.32
Acute phase
2 g (410 mg yield)	40.20±1.30^a*	48.43±1.08^a*	0.28±0.01^ab	7.66±0.82	3.13±0.24^a*b	176.00±2.07^a*
4 g (810 mg yield)	44.14±0.41^ab	48.90±1.01^a*	0.26±0.07^ab	5.86±0.01^a*	4.60±0.19^b*	147.40±2.26^a*
8 g (1570 mg yield)	41.80±0.11^ab	48.57±1.24	0.23±0.02^a**	8.31±1.92^a*	3.91±0.28	169.69±7.55^a*
Subchronic phase
2 g (410 mg yield)	39.47±1.41^a*	47.60±0.12^a*	0.46±0.09	8.09±0.86	3.83±0.12^a*	185.49±8.50
4 g (810 mg yield)	40.54±1.47	49.43±0.51^a*	0.20±0.07^a*	5.37±0.13^a*	3.91±0.24^a*	155.80±8.39
8 g (1570 mg yield)	44.09±2.07^a*	48.40±1.55	0.31±0.01^a*	8.11±1.05^a*	3.86±0.28	175.14±5.50

Values are expressed in mean±SEM.

Significantly different from baseline at 5% (P<.05).

a*b*

Significantly different from baseline and 10 days at 5% (P<.05).

a**b*

Significantly different from baseline at 1% (P<.01).

Discussion

In the present study, we found that ingestion of an infusion prepared from C. citratus leaf caused a significant increase in PCV and Hb at day 30 in male subjects of all groups, whereas a similar increase was found in female subjects treated with infusions prepared from 4 or 8 g of the leaf powder. Similarly, time- and dose-dependent significant increases in RBC counts were observed in male subjects treated with an infusion prepared from 4 or 8 g of C. citratus leaf powder for 10 and 30 days, and in female subjects treated with infusions prepared from 4 or 8 g for 30 days. Evidently, the aforestated results, as well as those from previous studies,^31,33,34 indicate that the extract, rather than inhibiting, enhanced the body's erythropoiesis system, including the kidney, which is the primary site for the production of erythropoietin (EPO), an acidic glycoprotein hormone that controls erythropoiesis.³⁵ This boosting effect could have been enhanced in part by the actions of the antioxidants present in the extract, which among others include saponins, flavonoids, polyphenols, tannins, and alkaloids. For instance, polyphenols present in C. citratus extract have been reported to increase the antioxidant activity and suppress markers of oxidative stress in experimental animals.³⁶ Recent studies have found that there is an increase in the activity of antioxidant enzymes (superoxide dismutase [SOD], catalase, glutathione peroxidase [GPx]) and a decrease in lipid peroxidase following the administration of C. citratus extract in animal model of experimentally induced oxidative stress.^37
–39 Oxidative stress is associated with increased vulnerability of RBCs to destruction and hence anemia. This could partly be due to increase autoimmune response, leading to RBC destruction, or by affecting RBC antioxidant enzymes (glutathione peroxidase and glutathione reductase). In their study, Waggiallah and Alzohairy⁴⁰ found a significant correlation between the deficiency of these enzymes and low Hb, RBC, and RBC indices (MCV, MCH, and MCHC) in diabetic subjects.

Glutathione peroxidase protects erythrocytes against intracellular or exogenous peroxides,⁴⁰ whereas glutathione reductase protects Hb, RBC enzymes, and biological cell membrane against oxidative damage by increasing the level of reduced glutathione (GSSGR) in the process of glycolysis.^41,42

Previous and present analysis of the bioactive constituents of C. citratus extract indicate that apart from the antioxidant effects of its phyto-actives, the extract also contains vitamins (A and C) and minerals (e.g., selenium [Se]) of known antioxidant activities.⁵ Several lines of evidence have shown that plasma concentrations of these vitamins may more adequately reflect dietary intake than tissue and body pool content.⁴³ Thus, the presence of these vitamins in the C. citratus infusion might have improved their total plasma concentrations. These 2 vitamins have been shown to scavenge reactive oxygen species (H₂O₂)⁴⁴ and increase the concentration of immune-active EPO in the perfusates of isolated perfused rat kidneys when added in combination with the perfusion medium.⁴⁵ For instance, vitamin C, one of the most important water-soluble antioxidants in C. citratus, is known to serve as the primary intracellular antioxidant in concert with glutathione.^43,46 It potentially increases intestinal iron absorption and iron metabolism from inert tissue stores with a resultant increase in transferrin saturation and Hb concentration. Vitamin C might also influence renal synthesis of EPO: in isolated rat kidneys, EPO production was enhanced in the presence of an antioxidative cocktail comprising vitamins A, C, and E,⁴⁵ and prooxidants reduced EPO synthesis in human hepatoma cells.⁴⁷ The antioxidant and hematopoietic induction potential of vitamin C supports its use in the management of several hematopoietic disorders associated with oxidative stress. According to Chris et al.,^48,49 vitamin C can overcome various forms of chemically and metabolically induced oxidative stress in human hemopoietic cell lines. JaJa et al. ⁵⁰ demonstrated a direct link between antioxidant activity and hemopoiesis boosting effect by showing that a vitamin C supplement, through its action as a free radical scavenger, significantly increased Hb levels in children suffering from sickle cell anemia. This could explain the absence of adverse effects on the hematopoiesis boosting system in subjects treated with C. citratus leaf extract, as demonstrated previously.⁵¹

Similarly, studies by others have shown that Se, an essential trace element present in C. citratus, is involved in several redox modulation activities leading to an increase in hematopoiesis.⁵² Evidently, Se functions as a redox switch in the form of many selenoproteins, including glutathione peroxidase (GSH-Px) and thioredoxin reductase.^52,53 It upregulates fork head transcription factor (FoxO3a), which is essential for the maintenance of the hematopoietic stem cell pool,⁵⁴ and mitigate oxidative threat posed during erythropoiesis by upregulating many antioxidant genes, including GSH-Pxi.^52,55 In their study, Kaushal et al. ⁵² observed a significant reduction in PCV, RBCs, Hb, and platelet counts in mice maintained on a diet deficient in Se for 8 weeks compared with those fed on either Se-adequate or Se-supplemented diets. A significant increase in the levels of carbonyl protein (indicator of raised free radicals and ROS levels in cells) was also found in the Se-deficient mice compared with Se-adequate mice. The erythrocytes of the Se-deficient mice also showed increased levels of Met Hb, signifying increased oxidation of ferrous ions in Hb. Their study findings suggested that erythrocytes from the Se-deficient group were more prone to hemolysis compared with those in the Se-adequate group. Obviously, the presence of Se in C. citratus extract could have contributed to improve RBC indices in the post-treatment group in the present study.

Aside from the aforenamed, C. citratus leaf extract also contain other hemopoiesis boosting vitamins and minerals, such as folic acid, niacin, pyridoxine, riboflavin, thiamine, calcium, copper, iron, magnesium, manganese, and zinc.^5,6,56 These have all been shown to boost the production of RBCs and other cell lineages. These findings are consistent with the observations made by Akah et al. ⁵⁷ who assessed the hematinic activity of a methanol extract of Brillantaisia nitens Lindau. In that study, the extract was found to contain vitamins B6, C, and E, as well as folic acid and iron. The authors attributed the increase in the hematologic indices to the direct effect of these vitamins and minerals present in the leaves of B. nitens.

These are well-known hematopoietic factors that directly influence the production of blood in the bone marrow. For instance, copper is also as essential as Fe for erythropoiesis,⁵⁸ and its deficiency leads to anemia due to decreased intestinal absorption of Fe in male and female rats. This ultimately causes a reduction in the number of blood variables, such as Hb and RBCs. Copper supplementation in defined doses corrects the resistance to EPO treatment in hemodialysis patients with anemia.⁵⁹ The nonsignificant increase in RBC indices (MCV, MCH, and MCHC) indicates that the RBCs were of normal morphology. This is probably because the extract has a rich supply of Fe, folate, and the B group vitamins. Deficiency of these vitamins and minerals manifest in structural abnormalities of RBCs. Thus, Fe deficiency causes RBCs to become microcytic and hypochromic, whereas folate deficiency results in a macrocytic form of RBCs. Although the erythropoiesis-modulated hemocytic variables (PCV, Hb, and RBC) increased in response to administration of the infusion, the WBC lineages decreased, with the exception of neutrophils and lymphocytes. This observation suggests that the C. citratus-mediated increase in PCV, Hb, and RBCs may be elicited through an effect on the stimulant cytokine EPO.⁶⁰ The selective increase in neutrophils and lymphocytes and decrease in platelet count in this study have been previously observed by other researchers.⁶¹ This action of the C. citratus leaf extract was found to be due to the presence of a bioactive constituent (eugenol), which displayed potent platelet inhibitory activity with an IC₅₀ of 46.6 μM, in comparison to aspirin with an IC₅₀ of 46.1 μM. This is thought to be due to the ability of eugenol to inhibit the formation of thromboxane A₂ and B₂, without any influence on the lipoxygenase pathway. Furthermore, eugenol, like aspirin, appears to inhibit the COX activity. This finding supports the use of C. citratus as a traditional Australian antiheadache medicine.⁶¹ In their study on pullet chicks, Adedeji et al. ⁶² observed that lymphocytes specifically proliferated, even though no significant increase occurred in the other WBC lineages. Ahumibe et al. ⁶⁰ also made similar observation in their study. In view of the major role that lymphocytes and neutrophils play in the immune and inflammatory mechanisms of the body in both man and animals, the decrease in platelets points to the antiplatelet activity of C. citratus, as demonstrated previously,⁶² and explains why C. citratus is contraindicated in patients with thrombocytopenia or those taking other antiplatelet drugs to prevent the risk of bleeding.

In conclusion, several evidences suggest that infusions prepared from C. citratus possess hematopoiesis boosting and, hence, antianemic effects, likely mediated through enhanced renal EPO synthesis, and suggest the presence of an antioxidative cocktail in the extract and its pharmacologic actions on the secondary causes of anemia. Thus, C. citratus extract may be a good candidate for antianemic drugs and may fulfill the holistic therapeutic needs of anemic patients due to its multicomponent and multitarget actions. These data support its use in traditional medicine for prevention and treatment of anemia.

Footnotes

Acknowledgments

The authors gratefully acknowledge the valuable assistance rendered by the paramedical and nonmedical staff in the Department of Physiology, University of Uyo, Nigeria.

Author Disclosure Statement

The authors have declared that no competing financial interests exist.

References

Gbadamosi

, Moody

, Yekini

: Nutritional composition of ten ethnobotanicals used for the treatment of anemia in southwest Nigeria. Eur J Med Plants, 2012; 2:140–150.

Kamatenesi-Mugisha

, Oryem-Origa

: Traditional herbal remedies used in the management of sexual impotence and erectile dysfunction in western Urganda. Afr Health Sci, 2005; 5:40–49.

Foote

, Cohen

: Medicinal herb use and the renal patient. J Ren Nutr, 1998; 8:40–42.

Okpuzor

, Adebesin

, Ogbunugafor

, Amadi

: The potential medical plants in sickle cell disease control: a review. IJBHS, 2008; 4:47–55.

Aftab

, Ali

, Aijaz

et al.: Determination of different trace and essential element in lemon grass samples by x-ray flouresence spectroscopy technique. IFR, 2011; 18:265–270.

USDA (2008). United States Department of Agriculture Research: Agriculture Research Services, Beltville; Gerplasm Resources Information. www.Nal.Usda.Gov/Fnic/Foodcomp/Search/ (accessed January 17, 2013 ).

Lorenzetti

: Myrcene mimics the peripheral analgesic activity of lemongrass tea. J Ethnopharmacol, 1991; 34:43–48.

de-Oliveira

, Ribeiro-Pinto

, Otto

, Goncalves

, Paumgartten

: Induction of liver monoxygenase by B-myrcene. Toxicology, 1997; 124:135–140.

Wannissorn

, Jarikasem

, Siriwangchai

, Thubthimthed

: Anti-bacterial properties of essential oils from Thai medicinal plants. Fitoterapia, 2005; 76:233–236.

10.

Nakagawa

, Mazzali

, Kang

, Kanellis

, Watanabe

, Sanchez-Lozada

, et al.: Hyperuricemia causes glomerular hypertrophy in the rat. Am J Nephrol, 2003; 23:2–7.

11.

Holetz

, Ueda-Nakamura

, Filho

, Cortez

, Morgado-Diaz

, Nakamura

: Effect of essential oil of Ocimum gratissimum on the Typanosomatid Herpetomonas samuelpessoai . Acta Protozool, 2003; 42:269–276.

12.

Puatonachokchai

, Kishida

, Denda

, Murata

, Konishi

, Vinitketkumnuen

: Inhibitory effects of lemon grass (Cymbogon citrates, stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats. Cancer Lett, 2002; 183:9–15.

13.

Abe

, Maruyama

, Hayama

, Inouye

, Oshima

, Yamaguchi

: Suppression of neutrophil recruitment in mice by geranium essential oil. Mediators Inflamm, 2004; 13:21–24.

14.

Masuda

, Odaka

, Ogawa

, Nakamoto

, Kuninaga

: Identification of geranic acid, a tyrosinase inhibitor in lemongrass (Cymbopogon citrates). J Agric Food Chem, 2008; 56:597–601.

15.

Mansour

, Newairy

, Yousef

, Sheweita

: Biochemical study on the effects of some Egyptian herbs in alloxan-induced diabetic rats. Toxicol, 2002; 170:221–228.

16.

Kumaran

, D'Souza

, Agarwal

, Bokkolla

, Balasubramaniam

: Geraniol, the putative anthelmintic principle of Cymbopogon martini . Phytother Res, 2003; 17:957.

17.

Tchoumbougnang

, Zollo

, Dagne

, Mekonnen

: Invivo antimalaria activity of essential oils from Cymbopogon citratus and Ocimum gratissimum on mice infected with Plasmodium berghei. Planta Med, 2005; 71:20–23.

18.

Negrelle

, Gomes

: Cymbopogon citrates (DC.) Stapf: chemical composition and biological activities. Rev Bras Pl Med Botucatu, 2007; 9:80–92.

19.

Oleyede

: Chemical profile and antimicrobial activity of Cymbopogon citratus leaf. J Nat Prod, 2009; 2:98–103.

20.

Shah

, Abramowitz

, Hostetter

, Melamed

: Serum bicarbonate levels and the progression of kidney disease: a cohort study. Am J Kidney Dis, 2009; 54:270–277.

21.

Trease

, Evans

: A Textbook of Pharmacognosy. 13th Ed., Baluire, Tindali, London, 1989, pp. 100–101.

22.

Sofowora

: Medicinal Plant and Traditional Medicine in Africa. 2nd Ed., Spectrum Books Ltd., Ibadan, Nigeria, 1993, pp 289.

23.

Association of Official Analytical Chemist (AOAC). 1990. Official Methods of Analysis. 15th Ed., Washington, DC, USA.

24.

Ismai

, Fun

: Determination of vitamin C, β-carotene and riboflavin content in five green vegetables originally grown. Mal J Nutr, 2003; 9:31–39.

25.

Klimes

, Jedlicka

: Determination of water- and fat-soluble vitamins in different matrices using high-performance liquid chromatography. Chem Pap, 2005; 59:202–222.

26.

Makkar

AOS

, Goodchild

: Quantification of Tannin: A Laboratory Manual. International centre for. Agricultural Research in the Dry area (ICARDA). Aleppo, Syria, 1996, pp. iv+25.

27.

Fasuyi

, Nonyerem

: Biochemical, nutritional and hematological implications of Telferia occidentalis leaf meal as protein supplement in broiler starter diets. Afr J Biotechnol, 2007; 6:1055–1063.

28.

Young

, Greaves

: Influence on variety and treatment of phytin contents of wheat. Food Res, 1940; 5:103–105.

29.

Moir

: The determination of oxalic acid in Plants. Queensl J Agric Sci, 1953; 10:1–3.

30.

Ernst

Herbal remedies for anxiety- a systematic review of controlled clinical trials. Phytomedicine, 2006; 13:205–208.

31.

Leite

, Seebra

, Maluf

, et al.: Pharmacology of lemongrass (Cymbopogon citratus stapf). Assessment of eventual toxic; hypnotic and anxiolytic effects on humans. J Ethnopharmacol, 1986; 17:75–83.

32.

Olaniyi

, Sofowora

, Oguntimehin

: Phytochemical investigation of some Nigerian plants used against fever. II. Cymbopogon citratus . Planta Med, 1975; 28:186–189.

33.

Omer

HAA

, Elallawy

HMH

, AbdEL-Samee

, Maghraby

: Productive performance of rabbits fed diets containing lemongrass or active dried yeast. Am Eur J Agric Environ Sci, 2010; 7:179–187.

34.

Hanisa

, Hadijah

, Rasedee

, Tarmizi

: Sub-acute oral administration of Cymbopogon citratus stem infusion and its effect on blood biochemical parameters, body and organ weight in rats. J Trop Agric Food Sci, 2011; 39:1–7.

35.

Noguchi

, Asavaritikrai

, Teng

, Jia

: Role of erythropoietin in the brain. Crit Rev Oncol Hematol, 2007; 64:159–171.

36.

Ojo

, Kabutu

, Bello

, Babayo

: Inhibition of paracetamol-induced oxidative stress in rats by extract of lemongrass (Cymbopogon citratus) and green tea (Camillia sinensis) in rats. Afr J Biotechnol, 2006; 5:1227–1232.

37.

Olorunsanya

, Olorunsanya

, Bolu

, Adejumobi

, Katode

: Effect of graded levels of lemongrass (Cymbopogon citratus) on oxidative stability of raw or cooked pork patties. Pak J Nutr, 2010; 9:467–470.

38.

Pei

, Mokhtar

, Mohammad

: Antioxidant potential of Cymbopogon citratus extract: alleviation of cabon tetrachloride-induced hepatic oxidative stress and toxicity. Hum Exp Toxicol, 2012; 31:81–91.

39.

Arhoghro

, Kpomah

: Cymbopogon citratus aqueous extract alleviates cisplatin-induced renal oxidative stress and toxicity in albino rats. Am J Res Comm, 2013; ISSN:2325–4076.

40.

Waggiallah

, Alzohairy

: The effect of oxidative stress on human red cells glutathione reductase level, and prevalence of anemia among diabetics. N Am J Med Sci, 2011; 3:344–347.

41.

Chang

, van der Hoeeven

, Haddox

: Glutathione reductase in the red blood cells. Ann Clin Lab Sci, 1978; 8:23–29.

42.

Johnson

, Goyette

, Ravindranath

, Ho

: Red cells from glutathione peroxidase-1-deficient mice have nearly normal defenses against exogenous peroxidase. Blood, 2000; 96:1985–1988.

43.

Lenton

, Sane

, Therriault

, Cantin

, Payette

, Wagner

: Vitamin C augments lymphocyte glutathione in subjects with ascorbate deficiency. Am J Clin Nutr, 2003; 77:189–195.

44.

: Cellular defense against damage from reactive oxygen species. Physiol Rev, 1994; 74:139–162.

45.

Jelkmann

, Pagel

, Hellwig

, Fandrey

: Effects of antioxidant vitamins on renal and hepatic erythropoietin production. Kidney Int, 1997; 51:497–501.

46.

Okano

, Masuda

, Narita

, et al.: Retinoic acid up-regulates erythropoietin production in hepatoma cells and vitamin A-depleted rats. FEBS Lett, 1994; 349:229–233.

47.

Neumcke

, Schneider

, Fandrey

, Pagel

: Effects of pro- and antioxidative compounds on renal production of erythropoietin. Endocrinol, 1999; 140:641–645.

48.

Chris

, Jenine

, Steven

, Manunya

, James

: Dietary anti-oxidant protects hematopoietic cells and improves animals' survival after total-body irradiation. Radial Res, 2008; 169:384–396.

49.

Meister

: Mitochondrial changes associated with glutathione deficiency. Biochim Biophys Acta, 1995; 1271:35–42.

50.

Jaja

, Ikotun

, Gbenebites

, Teniye

: Blood pressure, hematologic and erythrocyte fragility changes in children suffering from sickle cell anaemia following ascorbic acid supplementation. J Trop Paed, 2002; 48:4203–4206.

51.

Tarkang

, Agbor

, Tsabang

, et al.: Effect of long-term oral administration of the aqueous and ethanol leaf extract of Cymbopogon Citratus (DC. ex Ness) Stapf . Ann Biol Res, 2012; 3:5561–5570.

52.

Kaushal

, Hegde

, Lumadue

, Paulson

, Prabhu

: The regulation of erythropoiesis by selenium in mice. Antioxid Redox signal, 2011; 14:1403–1412.

53.

Allan

, Lacourciere

, Stadtman

: Responsiveness of selenoproteins to dietary selenium. Annu Rev Nutr, 1999; 19:1–16.

54.

Miyamoto

, Araki

, Naka

, et al.: Foxo3a is essential for maintenance of the hematopietic stem cell pool. Cell stem cell, 2007; 1:101–112.

55.

Nemoto

, Finkel

: Redox regulation of folkhead proteins through a p66shc-dependent signaling pathway. Science, 2002; 295:2450–2452.

56.

Asaolu

, Oyeyemi

, Olanilokun

: Chemical compositions, phytochemical constituents and invitro biological activity of various extracts of Cymbopogon citratus . Pakistan J Nutr, 2009; 8:1920–1922.

57.

Akah

, Okolo

, Ezike

: The heamatinic activity of methanol leaf extract of brillantasia nitens lindau (acanthaceae) in rats. Afr J Biochem, 2009; 8:2389–2393.

58.

Hermann

, Lewis

: The stimulating action of copper erythropoiesis. J Nutr, 1933; 6:465–472.

59.

Huguchi

, Yoshihiro

, Kazuyoshi

, et al.: Correction of copper deficiency improves erythropoietin unresponsiveness in hemodialysis patients with anaemia. Intern Med, 2006; 45:271–273.

60.

Ahumibe

, Braide

: Effect of gavage treatment with pulverized garcinia kola seeds on erythrocyte membrane integrity and selected hematological indices in male wistar rats. Niger J Physiol Sci, 2009; 24:47–52.

61.

Grice

, Rogers

, Griffiths

: Isolation of bioactive compounds that relate to the anti-platelet activity of Cymbopogon ambiguus. Evidence-Based Complement. Alternat Med, 2011, doi:10.1093/ecam/nep213

62.

Adedeji

, Favinu

, Ameen

, Olayeni

: Effect of dietary bitter kola (garcinic kola) inclusion on body weight, hematology and survival rate of pullet chick. J Anim Vet Adv, 2006; 5:184–187.