Platelet-Increasing Effects of Euphorbia hirta Linn ... Effects of Euphorbia hirta Linn. (Euphorbiaceae) in Ethanol-Induced Thrombocytopenic Rat Models Jovencio G. Apostol1,2,3, James

IJPFR, Jan-Mar 2012; 2(2):1-11 Original research article ISSN 2249 – 1112

Apostol et al © 2012 International Journal of Pharmaceutical Frontier Research

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http://www.ijpfr.com

Platelet-Increasing Effects of Euphorbia hirta Linn.

(Euphorbiaceae) in Ethanol-Induced Thrombocytopenic Rat

Models

Jovencio G. Apostol1,2,3

, James Viktor A. Gan1, Ryan Justin B. Raynes

1, Anna Andrea S.

Sabado1, Andrea Q. Carigma

1, Librado A. Santiago

1,2,3, Mafel C. Ysrael

1,2,3

Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas, España, Manila, Philippines

1

UST- Research Center for Natural and Applied Sciences2

The Graduate School, University of Santo Tomas, España, Manila, Philippines3

The antithrombocytopenic effect of the solution of the lyophilized decoction of Euphorbia hirta was

investigated in Sprague-Dawley rats with subnormal platelet counts induced by ethanol. E. hirta decoction prepared by

boiling the fresh entire plant in distilled water for 15 minutes was lyophilized and the resulting solid was dissolved in

distilled water and used to determine total phenolics and to prepare stock solutions administered to thrombocytopenic

rats. The polyphenolic contents of the lyophilized decoction determined by Folin-Ciocalteu and Fast Blue BB assays

revealed the presence of more reducing phenolics than reducing non-phenolics. The antithrombocytopenic activity of E.

hirta was assessed by platelet count, bleeding time and clotting time determination in rats randomized into four groups.

Group A served as the treated group given 100mg/kg of E. hirta. Group B served as the positive control group given

intraperitoneal ethanol at 3g/kg body weight and Group C and D were identified as the vehicle and time control groups,

respectively. Results showed a significant increase in the platelet count and decrease in bleeding and clotting times in

the ethanol-induced thrombocytopenic rats given E. hirta for 14 days. Furthermore, comparisons of the histopathologic

examination results of all groups indicated decreased liver sinusoidal dilation in the E. hirta treated rats. Therefore, the

potential use of E. hirta as an antithrombocytopenic decoction is attributable to its effect on platelet distribution and

possibly to the platelet protective activity of its antioxidant polyphenolic constituents.

Keywords: Euphorbia hirta, thrombocytopenia, platelet, bleeding time, clotting time, polyphenolics

*Corresponding Author Email: [email protected]

INTRODUCTION

Thrombocytopenia (TCP) is a condition wherein there is an abnormally low platelet count in the body

(usually less than 150 x 109/L). This condition may result from intake of drugs such as alcohol,

heparin, chloramphenicol, and cancer chemotherapeutic drugs as well as from various diseases states

such as chronic liver disease and dengue hemorrhagic fever. Current treatment for this condition

involves the use of expensive recombinant thrombopoetin and interleukin-11 to stimulate platelet

production. Platelet transfusions have also been used, but with only limited efficacy in restoring

platelet count. [1] Evidently there is still a need for a more affordable and convenient supportive

treatment for these patients.[2] Euphorbia hirta is an annual herb that belongs to the family

Euphorbiaceae also known as Spurge family. The plant has a hairy stem that is much-branched from

the base. These ascending branches may be simple or forked with a reddish or purplish color where

broad leaves are attached. [3] This plant thrives in open wastelands, grasslands, pathways, and

roadsides. [4] Tannins, flavonoids, phenolic acids, saponins, and amino acids have been isolated from



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leaves of the plant. [5] Other constituents isolated from the aerial parts of the plant include:

leucocyanidol, quercitol, camphol, quercetrin, dihydroellagitannins, and euphorbins. [6] Williams et

al. (1997) found that the stems and leaves of the E. hirta contained angiotensin converting enzyme

inhibitors using enzyme linked immunosorbent assay. [7] The fresh leaves of E. hirta are traditionally

used in the Philippines as a cure for dengue fever. Other folkloric uses include: as an anti-asthmatic

[4], antimicrobial [8-10], diuretic [11], and anti-inflammatory [12]. The plant has also been found to

exhibit a sedative-anxiolytic activity [13] and gastrointestinal motility stimulating properties (5).

Previous studies have reported the antioxidant activity of E. hirta [14,15]. The lyophilized aqueous

extract was found to be non-toxic when administered either IP or PO to mice at doses up to 100mg/Kg

[13]. The decoction of E. hirta is reported to increase platelet count when administered per orally,

however, substantial evidence about its effects on platelets and other blood components are limited.

[4] Since the plant’s reputation as a traditional treatment is based largely on anecdotal accounts of its

efficacy, health officials are cautious about recommending its use until a more concrete understanding

of the plant’s pharmacologic action is obtained. It is possible that the anti-thrombocytopenic effect is

due to inhibition of platelet oxidation because of the polyphenolic compounds in the crude extract [14,

15]

The chronic use of ethanol has been known to cause thrombocytopenia by altering platelet distribution

which would subsequently lead to the platelets being sequestered into the spleen. [16] As one of the

most popular substances used worldwide with a variety of commercial and industrial applications,

ethanol was used in this study for the induction of thrombocytopenia.

This study was conducted to determine the validity of the anti-thrombocytopenic effect of E. hirta in

animal models with subnormal platelet counts due to ethanol. The platelet count, bleeding time, and

clotting time was determined to assess this effect of the plant. Histopathological analysis of the liver

and spleen was conducted to obtain further evidence of the mechanism of E. hirta in

thrombocytopenia. The total phenolics of the plant were confirmed using the Folin-Ciocalteu Method

and Fast Blue BB Assay. This research is directed at providing evidence to substantiate the use of E.

hirta as a possible form of treatment for drug and disease-induced thrombocytopenia.

MATERIALS AND METHODS

Reagents and Equipment:

Reagent grade Folin-Ciocalteau reagent, sodium carbonate, and 95% ethanol was purchased from

Belman Laboratories (Philippines). Fast Blue BB reagent was from Ms. Marjorie B. Medina of the US

Department of Agriculture. Thermo-Heto Powder Dry LL 300 Freeze Dryer by Thermo Fisher

Scientific, (Pittsburgh, PA USA) was used. All substances used were reagent grade. Micros Counter

(Johnson & Johnson USA) was used to determine the platelet count. Genesys 10 UV/Vis

Spectrophotometer from Excellent Technology Co. (China) was used to take the absorbances.

Plant collection and Authentication:

Euphorbia hirta was collected from its natural habitat in San Luis, Pampanga, Philippines. A sample

specimen was declared authentic through a certificate (Control No. 240, OR No. 7079360) issued by

Dr. Wilfredo F.Vendivil of the National Museum of Philippines.

Preparation of Euphorbia hirta decoction:

A decoction was made by boiling 100 grams of fresh whole plant, previously washed of adhering soil

and debris, in 500 mL of water for 15 minutes at 100oC. The decoction was vacuum filtered, stored in

glass vials, and frozen for subsequent lyophilization. The percent yield was computed with the



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lyophilized extract. The lyophilized extract was then refrigerated for future use for the Phenolic

Content assay and treatment for the thrombocytopenic rats.

Determination of Total Phenolic Content:

Folin-Ciocalteu Method

The preparation method for this test followed a micronized procedure. [17] Stock solutions of 0.7 M

sodium carbonate and 10 gallic acid standard solutions of decreasing concentrations were prepared

ranging from 0.5 mg/mL decreasing to 9.7 x 10-1

µg/mL Twenty milligrams of lyophilized extract was

initially used in the serial dilution. Concentrations of the five stock solutions of extract were obtained

ranging from 2 mg/mL decreasing to 0.125 mg/mL.

50 microliters of GA standard or sample was transferred into borosilicate tubes which was followed

by the addition of 430 microliters of distilled water and 20 microliters of Folin-Ciocalteu reagent. The

resulting mixture was thoroughly mixed and saturated Na2CO3 (50 microliters) were added. An

additional 450 microliters of distilled water was added and was allowed to react for 1 hour. The

optical density was measured at 765 nm. This procedure was done in five replications.

The amount of phenolic compounds equivalents to gallic acid in E. hirta was determined by

comparing the absorbance of the sample to the gallic acid standard curve.

Fast Blue BB Method

Using a 100 ppm (µg/mL) gallic acid concentration, the spectrophotometric scan (200-800 nm) of

Fast Blue BB and gallic acid interaction was determined. Gallic acid (0.1 mL of 100 ppm) was

transferred to borosilicate tubes, followed by the addition of 0.1 mL Fast Blue BB (0.05 or 0.025%),

and mixed for 1 min. Saturated Na2CO3 (0.1 mL) was added, and the absorbance was scanned at 60

minutes reaction time. Blanks were also used to correct for non-phenolic constituents that may have

an optical density near the specified wavelength. This procedure was done in five replications.

Test Animals:

Animal protocol was done in accordance with Institutional Animal Care and Use Committee

guidelines. Approval of animal protocol was secured from the Philippine Department of Agriculture

Bureau of Animal Industry (Reference No AR-2011-022) before animal procurement.

Male Sprague-Dawley rats, weighing 180-200 grams were purchased from the University of the

Philippines Manila Department of Pharmacology. The animals were housed in the University of Santo

Tomas Research Center for the Natural and Applied Sciences Animal House for 7 days for

acclimation. The animals were maintained under the standard conditions of relative humidity

(55±5%), temperature (22-250C) and lighting sequence of 12 h light, 12 h dark phases. All animals

were fed standard rat pellet diet and given free access to water. Food and water were both replenished

daily. Experimental animals were handled in accordance with protocols approved by the University of

Santo Tomas Institutional Animal Care and Use Committee (UST-IACUC). The test animals were

divided into 4 groups (A, B, C, and D) with 6 animals in each group. Group A served as the test group

which was given the E. hirta decoction after thrombocytopenia induction, Group B was given ethanol

only and served as the positive control, Group C was the vehicle control which was given an equal

volume of water during the E. hirta administration, Lastly, Group D served as the time control which

did not receive any treatment during the course of the experiment.

Platelet Count Determination:

Blood was collected via the tail-tipping method. The tip of the tail was cleaned with alcohol and 2

mm was cut with sharp scissors. One drop of blood was allowed to flow and the second drop of blood



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was placed on a glass slide for the clotting time determination. Next, 0.5 mL of blood was collected in

EDTA tubes for platelet count on Micros Counter. A total of 4 blood collections were performed on

all test animals: before induction of thrombocytopenia (day 0), 7 days after induction of

thrombocytopenia (day 7), 7 days after E. hirta administration (day 14), and 14 days after E. hirta

administration (day 21).

Bleeding Time Determination:

Bleeding time was determined using Duke’s method with some modifications. The exposed tail tip

from the blood extraction was cleaned with distilled water and sterile gauze. The bleeding tip was

blotted on a #9 Whatman filter paper until no more blood was seen on the filter paper. The time from

first application to the disappearance of blood was recorded. This procedure was repeated during

every blood collection.

Clotting Time Determination:

The blood drop collected on the glass slide was rubbed using a lancet until fibrin threads were seen.

The time from first contact with lancet and the formation of threads was recorded. This procedure was

repeated during every blood collection.

Induction of Thrombocytopenia:

Groups A and B were used in the induction of thrombocytopenia. These groups were administered

with ethanol 3 g/ kg body weight intraperitoneally for 7 days. [16]

Euphorbia hirta Treatment :

Group A, after induction of thrombocytopenia by ethanol, was administered Euphorbia hirta

decoction at a dose of 100 mg/kg body weight by oral gavage. The decoction was given for fourteen

days and blood collections were done on the 7th

and 14th

days of treatment.

Histopathological Examination:

The test animals were killed by cervical dislocation after all relevant procedures. The liver and spleen

were isolated and sent to High Precision Diagnostics (Philippines) for slide preparation. Comparisons

between group A (Ethanol + E. hirta decoction), group B (Ethanol alone), and the time control (no

treatment) group were made.

All readings were done by a certified histopathologist and subject to his observations. The liver was

inspected for sinusoidal dilation, for dilation of the central vein, and for cloudy swelling. The findings

for sinusoidal dilation were categorized as 1+ (10-20%), 2+ (30-50%), or 3+ (>50%). The remaining

criteria were categorized as positive or negative.

Presence of sinusoidal dilation, hyperplasia with white pulp, and congestion with red pulp was

observed in the spleen. Sinusoidal dilation of the spleen was categorized similar to that of the liver.

Spleen hyperplasia was observed as positive or negative, and congestion of the spleen was ranked as

negative, 1+ (mild), 2+ (moderate), or 3+ (marked).

Statistical Treatment of Data:

All statistical analyses were performed at a 0.05 level of significance using SPSS 17.A dependent t-

test was performed to verify a significant decrease of platelet count, increase of bleeding and clotting

time after induction with ethanol. One-Way Analysis of Variance (ANOVA), with Tukey’s HSD test

as a post-hoc procedure, was used to determine if there exist significant differences in the E. hirta

treated, time, and vehicle control groups during the course of the treatment phase. A Kruskal-Wallis



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analysis of variance was used to compare the sinusoidal dilation of the liver and spleen and splenic

congestion while a generalized Fisher’s exact test (SAS 9.0) was used for the rest of the

histopathological analysis of the E. hirta treated, time, and vehicle control groups.

RESULTS AND DISCUSSION

Plant Decoction, Lyophilized Extract and Percentage Yield:

The choice of solvent and condition of plant material was made to emulate the traditional way of

preparing E. hirta by the native Filipinos. A decoction, made from fresh E. hirta plant, appeared as a

brown solution prior to vacuum filtration. After lyophilization, the end product appeared as a brown

powder. It was found that lyophilization preserves the activity of E. hirta. [18] The net yield was

computed to be 48% from the fresh plant material.

Abubakar (2009) stated that water is the best extracting solvent for E. hirta; however, the percentage

yield obtained in this study is much higher compared to his preparation which resulted in a net yield

of only 3.9% (3). The age of the plant and the condition of the plant material upon extraction is the

most likely cause of the big difference in the net yield as his study utilized dried plant material while

this study extracted from fresh E. hirta plant. It is to be noted that fresh E. hirta will yield more active

constituents than the dried plant. [18]

Total Phenolic Content:

A gallic acid standard curve was obtained by plotting concentration versus absorbance at 765 nm. The

curve yielded an R2 value of 0.9996 in Folin-Ciocalteu and 0.9987 in Fast Blue BB.

The milligram gallic acid equivalent (GAE) per 100 mg sample derived from the regression line is

1.92 ±0.06 expressed as 3.00% relative standard deviation from the Folin-Ciocalteu method and 2.54

± 0.09 expressed as 3.00% relative standard deviation from the Fast Blue BB method.

The results from the Folin-Ciocalteu assay indicate the reducing property of substances (i. e.

antioxidant potential). This method is based on the reduction of tungsten and molybdenum oxides

yielding a color change in E. hirta from yellow to blue. This blue color has a maximal absorption at

765 nm. The intensity of absorption is proportional to the concentration of polyphenolic compounds

(Table 1). The results from Fast Blue BB method is based on interactions of phenolics with Fast Blue

BB diazonium salt in alkali pH, forming azo complexes, with the absorbance measured at 200-800 nm

(Table 2). In most cases, Fast Blue BB would yield higher ratios of total phenolics as compared to the

Folin-Ciocalteau assay due to direct action with phenolics. [19, 20] Polyphenols have demonstrated

excellent antioxidant properties. [18]

TABLE 1 Total phenolics from E. hirta by Folin-Ciocalteu

Stock GAE SD GAE (mg) SD

Mg/mL Stock Stock 100mg Ex 100mg Ex % RSD

E. hirta 21.0 40.31 1.21 1.92 0.06 3.00

TABLE 2 Total phenolics from E. hirta by Fast Blue BB

Stock GAE (mg) SD GAE (mg) SD

Mg/mL Stock Stock 100mg Ex 100mg Ex % RSD

E. hirta 21.0 53.34 1.82 2.54 0.09 3.42 SD: Standard Deviation Ex: Extract

Olas et al. (2004) noted that induced platelet oxidation by peroxynitrite, a highly reactive oxidizing

species, causes the inhibition of platelet activation, reducing the different steps of platelet activation:

adhesion of platelets to collagen and fibrinogen, platelet aggregation and secretory process. It was



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found that peroxynitrite reacts with the thiol groups of the blood proteins. Furthermore, the prevention

of further oxidation was countered by the radical scavenging abilities of antioxidants, namely tea

polyphenolics and resveratrol. [21] In our study, the ratio (1.32) of polyphenolics obtained by Fast

Blue BB to Folin-Ciocalteu indicates the presence of more reducing phenolics than reducing non-

phenolics in E. hirta. [19] The effect of E. hirta is analogous to the application of antioxidants to

counter platelet oxidation and platelet dysfunction. By reducing platelet oxidation, platelet function

and lifespan are maintained, thus improving platelet count, bleeding and clotting times.

Effect of Euphorbia hirta on Platelet Count

Platelet counts were significantly decreased (p<0.05) after induction with ethanol. This decrease in

platelet count corresponds to the altered platelet distribution by ethanol which is responsible for the

development of thrombocytopenia. [22]

After 7 days of induction, there was a significant decrease in the platelet count in the rats given

ethanol. [840.00 ± 58.45 to 722.83 ± 43.65; t(df = 5)=4.082, p=0.010] compared to the baseline. This

13.3 ± 2.8% decrease in the platelet count was most likely caused by ethanol causing portal

hypertension and hypersplenism which resulted in thrombocytopenia. [23] These two factors cause

sequestration and accumulation of platelets in the spleen, decreasing the number of platelets available

in the periphery. Additionally, liver damage by ethanol can decrease production of thrombopoeitin

which decreases platelet production. [24, 25] E. hirta administration significantly increased platelet

count after 7 days [722.83 ± 43.65 to 982.83 ± 19.34; t(df=5)=5.128, p=0.004] by 38.9 ± 9.6%

compared to induction values. Continued administration until the 14th

day resulted in a significant

increase in platelets [722.83 ± 43.65 to 933.17 ± 95.38; t(df=5)=2.248, p=0.074] by 30.3 ± 13.9%

relative to induction values. Comparison between 7th

and 14th

day treatment values showed no

significant difference in the platelet count (Figure 1). The mean platelet count values of ethanol-

induced TCP rats did not significantly decrease from seventh day to 14 days after E. hirta treatment

indicating that the platelet count increase is sustained. This effect is attributable to improved platelet

redistribution from the spleen.

FIGURE 1 Platelet count comparison between ethanol induced rats and vehicle and time controls indicating significant

improvement (722.83 to 982.83109/L) after E. hirta administration(100mg/Kg BW/day) after 14 days. Results are

expressed as mean ± SEM.

Baseline 7 day Induction Treatment with E. hirta Treatment with E. hirta with Ethanol after 7 days after 14 days



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Effect of Euphorbia hirta on Bleeding and Clotting Time

Bleeding time is a parameter to assess hemostatic function. Hemostatic function is usually assessed

through the Duke’s method reflecting hemorrhagic potential. Upon induction with the ethanol,

bleeding times significantly increased [2.70 ± 0.37 to 6.22 ± 0.66; t(df = 5)=3.973, p=0.011] by 174.0

± 73%. The increase in bleeding time predisposes to prolonged hemorrhage and consequently

excessive blood loss. This directly correlates with the decrease in the number of platelets in

circulation, previously discussed. Upon administration of E. hirta, bleeding times significantly

improved by 49.5 ± 9.7% from induction values after 7 days [6.22 ± 0.66 to 2.97 ± 0.42;

t(df=5)=4.070, p=0.010], and improved by 59.38 ± 6.44% compared to induction values after 14 days

[6.22 ± 0.66 to 2.46 ± 0.37; t(df=5)=5.815, p=0.002]. The mean bleeding time of ethanol-induced

TCP rats did not differ from seventh day to 14 days (Figure 2). Improvement of bleeding time is

critical in hemorrhagic disorders such as DHF to reduce the number and severity of bleeding episodes.

FIGURE 2 Bleeding time comparison between ethanol-induced thrombocytopenic groups and vehicle and time

controls. Drug induced rats exhibited increased bleeding time which was decreased by E. hirta administration

(100mg/Kg BW/day) after 14 days (6.22-.2.46 minutes). Results are expressed as mean ± SEM.

Clotting time measures the degree of activation of the coagulation pathways. Unlike bleeding time

which measures hemostasis, clotting time assesses coagulation and the functionality of the clotting

factors. Clotting times significantly increased [0.83 ± 0.12 to 1.25 ± 0.15; t(df = 5)=6.041, p=0.002]

by 53.2 ± 10.5% after induction. These increases reflect the alteration of coagulation process and

clotting factor function by ethanol. Not only was the inducing agent able to affect platelet count, it

was also able to disrupt the coagulation process. These effects were reversed by 7 day administration

of Euphorbia hirta [1.25 ± 0.15 to 0.66 ± 0.15; t(df=5)=3.811, p=0.012] with improvements of 46.6 ±

11.3% from induction values, and on the 14th

day [1.25 ± 0.15 to 0.57 ± 0.10; t(df=5)=3.849,

p=0.012] of 50.8 ± 10.6%. The mean clotting time of ethanol-induced TCP rats did not significantly

decrease from seventh day to 14 days after E. hirta treatment (Figure 3).




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FIGURE 3 Clotting time comparison between ethanol-induced thrombocytopenic groups and vehicle and time controls.

Drug induced rats exhibited increased clotting time that was decreased by E. hirta administration (100mg/Kg BW/day)

after 14 days (1.25-0.57 minutes). Results are expressed as mean ± SEM.

Histopathological Examination

Tissue examination of the liver and spleen provided further evidence of thrombocytopenia as induced

by ethanol and the curative effects of the E. hirta decoction. Liver damage, which can result from

chronic intake of ethanol, leads to an increase in the blood pressure of the portal veins which will

impede the flow of blood out of the spleen. This leads to the platelets being sequestered in the spleen.

[26] The dilation of the sinusoids of these organs are often the result of poor venous outflow. [27]

The dilated sinusoids of the liver of the ethanol + E. hirta decoction group, the ethanol group, and the

control group were significantly different [p=0.031]. The ethanol + E.hirta decoction group differed

significantly from the ethanol only group [p = 0.012], but was similar to the control group [p = 0.317].

The ethanol only group was marked with dilations that are 2+ and 3+ while the ethanol + E. hirta

group and the control group only had the presence of 1+ and 2+ dilations (Table 3). Figures 4 and 5

shows the sinusoidal dilation of the ethanol + E. hirta group and the ethanol only group. Dilation of

the central vein of the liver and cloudy swelling of the liver were found to be similar for all three

groups [p=0.250].

TABLE 3 Degree of sinusoidal dilation of the liver in the different animal groups.

Groups Designation 1+ 2+ 3+ Median [IQR] p-value

1: Ethanol +

Euphorbia

Test group

(Group A)

25% 16.67% 0% 1+ [1+ to 2+]

0.031

2: Ethanol positive Postive Control

(Group B)

0% 16.67% 33.33% 3+ [2+ to 3+]

3: Control Time &

Vehicle

Control

(Group C&D)

0% 8.33% 0% 2+ [2+ to 2+]

Pairwise comparison: (1=3)<(2)




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FIGURE 4 Representative image of the liver of Sprague-Dawley rats (n=6; p-value=0.031) given ethanol only

showing dilated sinusoids (2+, 3+) and partial tissue necrosis.

FIGURE 5 Representative image of the dilated sinusoids (1+, 2+) of the liver of Sprague-Dawley rats

(n=6; p-value=0.031) given ethanol + E. hirta decoction

FIGURE 6 Representative image of the liver of the control group showing minimal sinusoidal dilation.



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Evidence of sinusoidal dilation was also found in the spleens of the test animals. White pulp is where

the T-cells and B-cells are located which provides an immune response to blood borne antigens. The

red pulp serves to filter blood constituents and to store red-blood cells and platelets [28]. Spleen

hyperplasia and congestions are the results of circulatory diseases which in this case is due to liver

damage. This malfunction of the normal processes of the organ leads to thrombocytopenia because of

the sequestration of platelets into the spleen. No notable difference was seen in the spleen sinusoidal

dilation [p=0.444], white pulp hyperplasia [p=0.748], and red pulp congestion [p=0.162] of the three

animal groups. This was most likely due to the short duration (1 week) of induction of

thrombocytopenia may have caused liver damage but was insufficient to cause any significant

changes to the spleen.

CONCLUSION

In conclusion, administration of 100mg/kg of the lyophilized decoction of E. hirta increased platelet

count in ethanol-induced thrombocytopenia after 7 days of administration. Continued administration

of the plant decoction resulted in the maintenance of this anti-thrombocytopenic effect. Improvement

of previously impaired hemostatic function is most likely due to the correction of altered platelet

distribution and sequestration caused by ethanol. Histopathological results, specifically liver

sinusoidal dilation, further corroborate these findings. Based on the results of the polyphenolic assays,

E. hirta contains more reducing polyphenols than non-polyphenolic compounds. Although this study

has established the hematologic effects of E. hirta relative to thrombocytopenia, further studies are

required to identify the polyphenols and relate their specific activity on platelets and platelet function.

FIGURE 7: Comparison between spleen tissue in Ethanol only group (top), in ethanol + E. hirta

decoction (middle), and control group (bottom). No notable differences were observed.



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ACKNOWLEDGEMENT

We are grateful for the generous support of the Philippine Department of Science and Technology-

Science Education Institute (DOST-SEI) and the invaluable suggestions of Dr. Rowen Yolo MD.

REFERENCES

1. Peeters K, Stassen JM, Collen D, Geet CV, Freeson K. Emerging treatments for

thrombocytopenia: Increasing platelet production. Drug Discovery Today 2008; 13:798-806.

2. http://www.ifrc.org/docs/appeals/rpts11/PHep14011102.pdf (accessed November 2, 2011).

3. Abubakar EM. Antibacterial activity of crude extracts of Euphorbia hirta against some

bacteria sssociated with enteric infections. Journal of Medicinal Plant Research

2009;3(7):498-505.

4. Quisumbing E. Medicinal Plants of the Philippines. Manila: Dept. of Agriculture & Natural

Resources; 1951.

5. Hore SK, Ahuja V, Mehta G, Kumar P, Pandey SK, Ahmad AH. Effect of Aqueous

Euphorbia hirta Leaf Extract on gastrointestinal motility. Fitoterapia 2006; 77. 35-38.

6. Sudakar M, Rao C, Rao P, Raju D, Venkateswarlu Y. Antimicrobial activity of Caesalpinia

pulcherrima, Euphorbia hirta and Asystasia gangeticum. Fitoterapia 2006; 77: 378-380

7. Barbosa JM, Martins V, Rabelo L et al. Natural products inhibitors of the angiotensin

converting enzyme (ACE). A review between 1980-2000. Re.bras.farmacogn 2006; 16(3)

8. Kumar OA, Naidu LM, Rao KG. Antibacterial Evaluation of Snake Weed (Euphorbia hirta

L.) Journal of Phytology 2010; 2:2075-6240.

9. Ogbulie JN, Ogueke CC, Okoli IC, Anyanwu BN. Antibacterial Activities and Toxicological

Potentials of Crude Ethanolic Extracts of Euphorbia hirta. African Journal of Biotechnology

2007; 6:1544-1548.

10. Vijaya K, Anathan S, Nalini R. Antibacterial Effect of theaflavin, polyphenon 60

(Camilliasinensis) and Euphorbia hirta on Shigella spp. – a cell culture study. Journal of

Ethnopharmacology 1995; 49:115-118.

11. Johnson PB, Abdurahman EM, Tiam EA, Abdu-Aguy I, Hussaini IM. Euphorbia hirta leaf

extracts increase urine output and electrolytes in rats. Journal of Ethnopharmacology 1995;

65:63-69.

12. Youssouf MS, Kaiser P, Tahir M et al. Anti-anaphylactic Effect of Euphorbia hirta.

Fitoterapia 2007; 78:535-539.

13. Lanhers MC, Fleurentin J, Cabalion P, Rolland A, Dorfman P, Misslin R, Pelt JM. Behavioral

Effects of Euphorbia hirta L: Sedative and Anxiolytic Properties. Journal of

Ethnopharmacology 1990; 29: 189-198.

14. Basma AA, Zakaria Z, Latha LY, Sasidharan S. Antioxidant activity and phytochemical

screening of the methanol extracts of Euphorbia hirta L. Asian Pacific Journal of Tropical

2011;4:386-390. 15. Sharma NK, Prasad R. Oxidative injury to protein and their protection by phenolic acid

antioxidants from Euphorbia hirta leaves. Journal of Biotechnology (2008); 136:720

16. Littleton J, Fenn CG, Umney ND, Yazdanbakhsh M. Effect of Ethanol administration on

platelet function in the rat. Alcoholism: Clinical and Experimental Research (2008); 6(4):

512-519.

17. Waterhouse AL. Determination of Total Phenolics. In Wrolstad R, Acree TE, Decker EA,

Penner MH, Reid DS, Schwartz SJ et al. eds. Current Protocols in Food Analytical

Chemistry (Supplement 6). John Wiley & Sons, Inc; 2002: 463-482.

18. Duez P, Livaditis A, Guissoi PI, Sawadogo M, Hanocq M. Use of an Amoeba proteus model

for in vitro cytotoxicity in phytochemical research. Application to Euphorbia hirta extract.

Journal of Ethnopharmacology 1991; 34:235-246.



12

19. Medina M. Determination of the total phenolics in juices and superfruits by a novel chemical

method. Journal of Functional Food 2011;3: 79-87.

20. Lester G, Lewers K, Medina M, Saftner R. Comparative analysis of strawberry total

phenolics via Fast Blue BB vs. Folin-Ciocalteu: Assay interference by ascorbic acid. Journal

of Food Composition and Analysis 2012; 27:102-107.

21. Olas B, Nowak P, Kolozijczyk J, Wachowicz B. The Effects of Antioxidants on

Peroxynitrite-induced Changes in Platelet Proteins. Thrombosis Research 2004; 113:399-

406.

22. Rodak BF, Fritsma G A, Doig K. Hematology: Clinical Principles and Applications.

Singapore: Elsevier; 2009.

23. Afdhal N, McHutchison J, Brown J et al. Thrombocytopenia Associated with Chronic Liver

Disease. Journal of Hepatology 2008; 48:1000-1007.

24. Salem RO, Laposata M. Effects of Alcohol on Hemostasis. American Journal of Clinical

Pathology 2005; 123:96-105.

25. Ballard HS. The Hematological Complications of Alcoholism. Alcohol Health & Research

World 1997; 21(1):42-52.

26. Brunicardi FC, Andersen DK, Biliar TR et al. Schwartz’s Principles of Surgery. McGraw-

Hill Professional; 2009.

27. Kakar S, Kamath P, Burgart L. Sinusoidal Dilatation and Congestion in Liver Biopsy.

Archives of Pathological and Laboratory Medicine 2004; 128:901-904.

28. Vanhoenacker FM, Beeck B, De Schepper AM, Salgado R., Scnoeckx A, Parizel PM.

Vascular Diseases of the Spleen. Seminars in Ultrasound, CT, and MRI 2007; 28(1): 35-51.

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