The Effect of Lyophilized Zingiber officinale (Ginger) Extract on the Quantitative
Platelet Count
Jose Edwardo Romero- Mamaat, MD, RMT1 *, Jan Michael A. Enriquez2, Carey Rachelle G.
Lim, Roxanne Mae S. Mailed, Sam Kevin C. Saclayan #, Luigi Albert S. Viquiera
1
Program Head, Department of Medical Technology, Far Eastern University, Manila,
Philippines
2
Bachelor of Science in Medical Technology, Far Eastern University, Manila, Philippines
* jemamaat@feu.edu.ph/ jeurisch@yahoo.com, # sam_kevin0328@yahoo.com
Abstract
This study is a quasi-experimental research that determined the in vitro antiplatelet activity of
lyophilized Zingiber officinale (Ginger) extract by inhibiting platelet aggregation through the
inhibition of Thromboxane A 2 synthesis. Ginger extract was lyophilized and was added to
blood samples with EDTA and platelet count was done manually using Rees and Ecker method
and results were compared with control and to specimen incubated at 37˚C. This study aimed
to determine the significant difference between the results of Quantitative platelet count using
EDTA and EDTA with lyophilized ginger extract and Quantitative platelet count using EDTA
with lyophilized ginger extract and EDTA with lyophilized ginger extract incubated at 37.5 C
for 5 minutes. Paired T test was employed and showed equal and unequal variance of 13.917
at df of 62 and 48.462 (p value of .000) between control and blood with EDTA; and lyophilized
ginger respectively; showing significant result. Meanwhile between blood specimen with
EDTA and lyophilized ginger and specimen that was incubated at 37.5 C showed a t value of
1.874, df of 62 and 59.745 at a p value of 0.066; which showed statistically insignificant
finding. In this study, Zingiber officinale has demonstrated antiplatelet property as evidenced
by the significant decrease in the platelet count among the samples and supported by the
calculated df. There was a significant difference seen in between the sample in EDTA and in
EDTA with lyophilized ginger stating that a decrease in the platelet count of the sample occurs
in EDTA with lyophilized ginger compared to the sample in EDTA only. However, there was
no significant difference in the platelet count of the sample in EDTA with lyophilized and the
sample in EDTA with lyophilized ginger incubated at 37°C for 5 minutes. Hence, regardless
of the environment, ginger has the ability to inhibit cyclooxygenase resulting to decrease of
platelet count.
Keywords: Zingiber officinale, lyophilized, EDTA, Thromboxane A 2
Introduction
Ginger, the rhizome of Zingiber officinale, is one of the most widely used species of
the ginger family (Zingiberaceae). The major pungent compounds found in ginger yielded
potentially active gingerols, which can be converted to shogaols, zingerone, and paradol. The
compounds 6-gingerol and 6-shogaol have shown a number of pharmacological activities, such
as antipyretic, analgesic, antitussive, and hypotensive properties. Ginger extract inhibits
platelet aggregation and thromboxane synthesis in vitro, leading to a medically significant
concern of bleeding tendencies. (Thorne, 2003)
This study is a quasi-experimental research done to test and evaluate the antiplatelet
property of the lyophilized ginger (Zingiber officinale) extract as compared to the standard
anticoagulant used for platelet counting. This study determined the antiplatelet property of the
lyophilized Zingiber officinale (ginger) extract; specifically determining the significant
difference between the results of the Quantitative platelet count (Rees and Ecker method) using
EDTA and EDTA with lyophilized ginger extract; and the significant difference between the
results of the Quantitative platelet count using EDTA with lyophilized ginger extract and
EDTA with lyophilized ginger extract incubated at 37°C for 5 minutes.
The results of this study posed its significance to the medical technologists, physicians, and
to persons with cardiovascular diseases and thrombocytopenia.
Methodology
This research utilized the following methods: preparation of ginger extract,
lyophilization, blood collection, specimen preparation, and quantitative platelet counting (Rees
and Ecker method).
The commercially available ginger was obtained from the local market and was brought
to the National Museum in Manila for authentication. The rhizome of ginger was washed,
peeled and cut into pieces. After cutting, the ginger rhizomes were grinded and made into paste.
The ginger paste was then placed on a cheese cloth was squeezed to produce its natural juice.
The juice obtained was stored in the refrigerator until ready for lyophilization. After the
lyophilization, the ginger extract was subjected to phytochemical analysis performed at
National Institute of Health at the University of the Philippines - Manila.
The process of lyophilization was initiated with the vials that were aseptically filled
with the solution or juice to be frozen dried and partially stopped with a special rubber closure
that allowed the escape of water vapor. The vials were then transferred to the freeze drier. Trays
of product were placed on shelves containing internal channels allowing circulation of silicone
oil or another heat transfer fluid. The product was first frozen to a low temperature to allow
complete solidification of the content of each vial. The chamber was evacuated until the
pressure was less than the vapour pressure of ice at the temperature of the product. After the
pressure had been reached, heat was applied to the shelves to provide the energy required for
the sublimation of ice. When the product was already dried, the vials were stoppered in place
within the drier by hydraulic compression of the shelf stack, pushing the stoppers to the fully
inserted positions.
Blood was extracted from subjects through venipuncture that
commenced with proper assembly of equipment (needle and syringe or EDTA tube) used.
Aseptic technique on the part of the phlebotomist was observed. This was followed by proper
identification and preparation of the subject. The phlebotomist selected the site, preferably at
the antecubital area (i.e. the bend of the elbow). Once the site for collection had been
determined, antiseptic like alcohol or tincture of Iodine Povidone was applied. This was
followed by the application of tourniquet, about 4–5 finger widths above the selected
venipuncture site. Apply the needle of the syringe swiftly into the vein at a 30 degree angle.
Once sufficient blood had been collected, the tourniquet was released while continuous
aspiration of blood was realized by pulling the plunger. Once the needed blood was collected,
a dried cotton was placed over the venipuncture site while the needle/syringe was being swiftly
withdrawn. The blood sample was then transferred to a test tube with purple stopper containing
Ethylene Diamine Tetra acetic Acid. Proper labeling of test tube and disposal of sharp materials
were also observed. Two milliliter of the anticoagulated blood was transferred to a sterile plain
tube and was added with 100 mg of lyophilized ginger extract. The aliquot was then mixed
until homogenous. The aliquot specimen also served as the specimen for quantitative platelet
counting using the Rees and Ecker method. The count obtained was then compared to the
platelet counts of control specimen without lyophilized ginger and to the platelet count of the
aliquot specimen that was heated to 37.5 C.
Rees and Ecker method: Rinse RBC pipette first with Rees ad Ecker’s fluid by
sucking in and out the diluting fluid. Suck blood (with EDTA or EDTA with ginger) to 0.5
mark of the RBC pipette. Suck diluting fluid up to the 101 mark. Shake the pipette for 3-5
minutes. Discard the first few drops. Charge the counting chamber and let it stand for at least
3 minutes. Count the platelets in the 4 corner large squares.
𝑃𝑙𝑡.𝐶𝑜𝑢𝑛𝑡𝑒𝑑 ×10×200
Computation:
𝑃𝑙𝑡. 𝑐𝑡.⁄𝑐𝑢. 𝑚𝑚. =
4
Normal Value: 140,000 – 340, 000/cu.mm
This study was conducted at Far Eastern University Manila utilizing 32 senior
students as subjects that were purposively selected.
Results:
Table 1. Quantitative Platelet Count of Subjects’ Blood Sample with EDTA, and Quantitative Platelet Count of
Blood Samples with EDTA and Lyophilized Ginger Extract
Quantitative Platelet Count (per mm3)
Patient No.
EDTA
EDTA w/ Ginger Extract
Trial 1
Trial 2
Trial 3
AVE
Trial 1
Trial 2
Trial 3
AVE
001
254,000
270,000
276,000
266,667
104,000
110,000
112,000
108,667
002
176,000
178,000
164,000
172,667
110,000
114,000
118,000
114,000
003
280,000
300,000
292,000
290,667
114,000
118,000
114,000
115,333
004
210,000
220,000
280,000
236,667
118,000
122,000
128,000
122,667
005
300,000
320,000
314,000
311,333
120,000
122,000
118,000
120,000
006
274,000
256,000
260,000
263,333
82,000
84,000
78,000
81,333
007
244,000
270,000
254,000
256,000
130,000
122,000
126,000
126,000
008
344,000
364,000
368,000
358,667
110,000
114,000
114,000
112,667
009
256,000
270,000
272,000
266,000
146,000
150,000
156,000
150,667
010
250,000
256,000
260,000
255,333
162,000
144,000
158,000
154,667
011
166,000
180,000
168,000
171,333
110,000
96,000
104,000
103,333
012
220,000
214,000
206,000
213,333
134,000
122,000
138,000
131,333
013
178,000
210,000
200,000
196,000
116,000
100,000
112,000
109,333
014
248,000
228,000
246,000
240,667
160,000
164,000
172,000
165,333
015
200,000
220,000
216,000
212,000
86,000
94,000
80,000
86,667
016
226,000
240,000
234,000
233,333
90,000
102,000
98,000
96,667
017
210,000
202,000
206,000
206,000
98,000
100,000
102,000
100,000
018
190,000
220,000
196,000
202,000
80,000
78,000
82,000
80,000
019
202,000
206,000
202,000
203,333
98,000
102,000
106,000
102,000
020
240,000
254,000
258,000
250,667
136,000
152,000
140,000
142,667
021
202,000
212,000
204,000
206,000
148,000
156,000
146,000
150,000
022
300,000
254,000
276,000
276,667
126,000
132,000
118,000
125,333
023
292,000
284,000
304,000
293,333
120,000
108,000
124,000
117,333
024
206,000
218,000
212,000
212,000
96,000
120,000
120,000
112,000
025
278,000
264,000
272,000
271,333
102,000
120,000
108,000
110,000
026
350,000
332,000
374,000
352,000
180,000
190,000
178,000
182,667
027
238,000
246,000
234,000
239,333
96,000
94,000
96,000
95,333
028
250,000
238,000
238,000
242,000
148,000
162,000
154,000
154,667
029
258,000
256,000
266,000
260,000
120,000
110,000
116,000
115,333
030
206,000
212,000
214,000
210,667
102,000
106,000
108,000
105,333
031
266,000
274,000
276,000
272,000
96,000
92,000
90,000
92,667
032
210,000
200,000
202,000
204,000
90,000
88,000
94,000
90,667
Table 1 shows the quantitative platelet count of the subjects’ blood samples with
EDTA as control specimen, as compared to the quantitative platelet count of samples with
EDTA and lyophilized ginger extract. Quantitative plate count shows a more specific detail
when it comes to the count of a patient’s platelet compared to the qualitative platelet count. In
this set of data, results showed that the platelet count of the subjects have an abrupt decrease
when the blood samples were added with lyophilized ginger extract compared to the control
blood samples.
Table 2. T-TEST: EDTA vs. EDTA with Ginger Extract
Result
t statistics df
p-value1
13.917
62
Equal Variance
.000
48.462 .000
Unequal Variance 13.917
Mean Difference
127208.313
127208.313
Lower Limit
108937.135
108835.058
Upper Limit
145479.490
145581.567
Table 2 shows the difference between the platelet count of the sample with EDTA,
and sample with EDTA and lyophilized ginger extract is statistically significant at the pvalue1 < 0.05. there is sufficient evidence that there is a significant difference between the
sample with EDTA, and the sample with EDTA and lyophilized ginger extract.
Table 3 Quantitative platelet count of the patients’ blood sample with EDTA and ginger extract, and with
EDTA and ginger extract incubated for 5 minutes at 37.5°C
Quantitative Platelet Count (per mm3)
Patient
EDTA w/ Ginger Extract
Incubated (5 mins. at 37°C)
No.
Trial 1
Trial 2
Trial 3
AVE
Trial 1
Trial 2
Trial 3
AVE
001
104,000 110,000 112,000 108,667 88,000
82,000
86,000
85,333
002
110,000
114,000
118,000
114,000
76,000
84,000
78,000
79,333
003
114,000
118,000
114,000
115,333
78,000
84,000
88,000
83,333
004
118,000
122,000
128,000
122,667
110,000
120,000
128,000
119,333
005
120,000
122,000
118,000
120,000
82,000
88,000
88,000
86,000
006
82,000
84,000
78,000
81,333
80,000
88,000
86,000
84,667
007
130,000
122,000
126,000
126,000
118,000
120,000
116,000
118,000
008
110,000
114,000
114,000
112,667
94,000
100,000
92,000
95,333
009
146,000
150,000
156,000
150,667
132,000
126,000
136,000
131,333
010
162,000
144,000
158,000
154,667
126,000
126,000
128,000
126,667
011
110,000
96,000
104,000
103,333
132,000
108,000
120,000
120,000
012
134,000
122,000
138,000
131,333
128,000
140,000
130,000
132,667
013
116,000
100,000
112,000
109,333
96,000
100,000
98,000
98,000
014
160,000
164,000
172,000
165,333
142,000
154,000
146,000
147,333
015
86,000
94,000
80,000
86,667
74,000
80,000
72,000
75,333
016
90,000
102,000
98,000
96,667
90,000
100,000
94,000
94,667
017
98,000
100,000
102,000
100,000
88,000
80,000
90,000
86,000
018
80,000
78,000
82,000
80,000
138,000
132,000
138,000
136,000
019
98,000
102,000
106,000
102,000
96,000
100,000
94,000
96,667
020
136,000
152,000
140,000
142,667
94,000
108,000
126,000
109,333
021
148,000
156,000
146,000
150,000
132,000
128,000
120,000
126,667
022
126,000
132,000
118,000
125,333
78,000
84,000
118,000
93,333
023
120,000
108,000
124,000
117,333
100,000
102,000
112,000
104,667
024
96,000
120,000
120,000
112,000
94,000
98,000
96,000
96,000
025
102,000
120,000
108,000
110,000
98,000
108,000
104,000
103,333
026
180,000
190,000
178,000
182,667
148,000
154,000
162,000
154,667
027
96,000
94,000
96,000
95,333
118,000
120,00
128,000
123,000
Quantitative Platelet Count (per mm3)
Patient
No.
EDTA w/ Ginger Extract
Incubated (5 mins. at 37°C)
Trial 1
Trial 2
Trial 3
AVE
Trial 1
Trial 2
Trial 3
AVE
028
148,000
162,000
154,000
154,667
118,000
128,000
126,000
124,000
029
120,000
110,000
116,000
115,333
98,000
110,000
108,000
105,333
030
102,000
106,000
108,000
105,333
92,000
98,000
92,000
94,000
031
96,000
92,000
90,000
92,667
88,000
90,000
94,000
90,667
032
90,000
88,000
94,000
90,667
106,000
110,000
112,000
109,333
Table 3 shows the quantitative platelet count of the subjects’ blood sample with
EDTA and lyophilized ginger extract, and quantitative platelet count of blood samples with
EDTA and ginger extract incubated for 5 minutes at 37.5°C. Decrease in the platelet count of
the patients were seen when the blood sample was incubated at 37.5°C. This shows that
decrease in platelet count is adverse when incubation is done, signifying that ingestion of
ginger may cause higher risk of causing a significant decrease in the platelet count.
Table 4. T-TEST: EDTA with ginger vs. EDTA with ginger (Incubated at 37°C)
Mean
Result
t statistics df
p-value1
Lower Limit
Difference
Equal
1.874
62
10760.469
-719.562
.066
Variance
Unequal
1.874
59.745 .066
10760.469
-728.187
Variance
Upper Limit
22240.500
22249.124
Table 4 shows the difference between the platelet count of the sample with EDTA
and lyophilized ginger extract, and EDTA with lyophilized ginger extract incubated @ 37°C
for 5 minutes is statistically insignificant at the p-value1 ≥ 0.05. There is sufficient evidence
that there is no significant difference between the sample in EDTA with lyophilized ginger
extract, and EDTA with lyophilized ginger extract incubated @ 37°C for 5 minutes.
Discussion and Conclusion
The study aimed to assess the anti-platelet activity and effectiveness of Ginger
(Zingiber officinale). Similar studies in the past were conducted and showed the ability of
Zingiber officinale to inhibit platelet aggregation by inhibiting the activity and synthesis of
thromboxane A2. According from different authors, the major components of Zingiber
officinale are gingerols, shogaols, zingerone, and paradols which have been shown to inhibit
arachidonic acid that induced human platelet aggregation.
This study utilized 3 different samples (blood with EDTA, blood with EDTA and
lyophilized ginger extract, and incubated blood with EDTA and lyophilized ginger extract)
using 100 milligram of lyophilized ginger and 2 ml of blood. The blood samples were
collected through venipuncture procedure. Quantitative platelet count was done using Rees
and Ecker method and the counts were compared and treated using T test.
From the result obtained, it can be concluded that ginger has an antiplatelet property
as evidenced by the significant decrease of platelet count between the control and
experimental group with anticoagulated blood with lyophilized ginger. This is accounted to
the effect of gingerol to cyclooxygenase; inhibiting the production of the by-product of
arachidonic acid, thromboxane A2, which is known to initiate platelet activation and
adhesion.
A significant difference was seen in between the sample in EDTA and in EDTA with
lyophilized ginger suggesting that there is a decrease in the platelet count of the sample in
EDTA with lyophilized ginger compared to the sample in EDTA only.
However, there was no significant difference in the plate count of the sample in
EDTA with lyophilized and the sample in EDTA with lyophilized ginger incubated at 37.5°C
for 5 minutes implying that the decrease of the platelet count from the both sample is not
remarkably important. Hence, it also suggests that heat may not significantly contribute to the
action of ginger in inhibiting cyclooxygenase thus resulting to decrease of platelet count.
The crude extract of ginger may be used as a natural alternative for antiplatelet or as a
potential platelet inhibitor for it has fewer side effects compared to commercially available
drugs. Due to lack of access to some facilities and equipment, in vivo testing was not
employed in the study. The researchers recommend that the study to be continued and be
utilized for in vivo testing to prove its effect on human body system. The researchers also
recommend that the future researchers use Brecker-Cronchite method instead of Rees and
Ecker’s method for the manual platelet counting.
References
1.Auta, K. (2011). Antimicrobial Properties of the Ethanolic Extracts of Zingiber officinale (Ginger) on
Escherichia coli and Pseudomonas aeruginosa . 37-39.
2.Baume L.Ac., A. (2009). Maintain Your Health with Ginger. Natural News.
Development and validation of an International Prognostic Score of thrombosis in World Barbui, T. (2012).
Development and validation of an International Prognostic Score of thrombosis in World Health Organization–
essential thrombocythemia . Blood .
3.Foster, S. (n.d.). GINGER ZINGIBER OFFICINALE. Your food is your medicine .
4.Geiger, J. L. (2005). The essential oil of ginger, Zingiber officinale, and anesthesia. The International Journal
of Aromatherapy , 7-14.
5.Grant, K. L. (2000). Alternative Therapies: Ginger. Medscape Today .
6.Grzanna R, L. L. (n.d.). Ginger--an herbal medicinal product with broad anti-inflammatory actions.
ncbi.govmed .
7.Grzanna R, L. L. (2005). Ginger--an herbal medicinal product with broad anti-inflammatory actions.
8.Heiss, e. (2010). Flavonols and Cardiovascular Disease Prevention.
9.Henein, N. R. (2007). Ginger (Zingiber officinale Roscoe): a hot remedy for cardiovascular disease?
10.Hong, N. (2008). Analysis of anti-platelet aggregation components of Rhizoma. Chin Med , 1226-1229.
11.Hong, N. (2008). Analysis of anti-platelet aggregation components of Rhizoma Zingiberis using chicken
thrombocyte extract and high performance liquid chromatography. Chin Med , 1226-1229.
12.Lam, N. Y. (2007). EDTA Is a Better Anticoagulant than Heparin or Citrate for Delayed Blood Processing
for Plasma DNA Analysis. 256-257.
13.Liao, Y.-R. e. (2012). Anti-Platelet Aggregation and Vasorelaxing Effects of the Constituents of the
Rhizomes of Zingiber officinale. Molecules .
14.Liao, Y.-R. e. (2012). Anti-Platelet Aggregation and Vasorelaxing Effects of the Zingiber officinale.
Molecules , 8929- 8933.
15.M.J. Silver, J. S. (n.d.). Arachidonic acid-induced human platelet aggregation and prostaglandin formation. .
16.Malhotra, S. (2003). Medicinal Properties of Ginger. Natural Product Radiance .
17.Mishra, R. K. (2012). Pharmacological Activity of Zingiber Officinale. INTERNATIONAL JOURNAL OF
PHARMACEUTICAL AND CHEMICAL SCIENCES .
18.Pancho, L. R. (2009). Reversed Effects between Crude and Processed Ginger Extracts. Short
Communications .
PhD, W. A. (n.d.). Herbal medication: potential for adverse interactions with analgesic drugs.
19.PL, B. (2003). Laboratory methods in hemostasis in Thrombosis and Hemorrhage. Philadelphia: Lippincott
Williams & Wilkins.
20.Prasad, e. (2012). To Investigate the action of Ginger-juice Zingiber Officinale Roscoe (ZINGIBERACEAE)
on blood coagulation process.Properties of Ginger. (n.d.). botanical-online .
21.Pryor, K. (n.d.). Review of Select Natural Blood Thinners.
22.Ramji, D. (2007). Isolation of Gingerols and Shogaols from Ginger and Evaluation of Their
Chemopreventive Activity on Prostate Cancer Cells and Anti-inflammatory Effect on 12-Otetradecanoylphorbol-13-acetate-induced Mouse Ear Inflammation.
23.Rodak, B. (2012). Hematology, Clinical Principles and Applications.
24.Tchombe, L. e. (2012). Therapeutic Effects of Ginger (Zingiber officinale). ISESCO Journal of Science and
Technology , 64-69.
25.Walters, S. (2009). Find Health and Healing Properties in Ginger. Natural News .
26.Wu, H. (2007). Isolation and Characterization of Natural Products from Ginger and Allium Ursinum.
27.Young, H.-Y. (n.d.). Synergistic Effect of Ginger and Nifedipine on Human Platelet Aggregation: A Study in
Hypertensive Patients and Normal Volunteers. 545-551.