International Journal of Research in Pharmacy and Pharmaceutical Sciences

International Journal of Research in Pharmacy and Pharmaceutical Sciences

ISSN: 2455-698X
Impact Factor: RJIF 5.22
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Volume 3; Issue 1; January 2018; Page No. 154-159

Evaluation of the analgesic property of the semi-purified flavonoids from the fronds of Diplazium
esculentum ethanolic extract
Cristal Mae B Pedong, Mary Regielynn P Ilao, Sarah L De Lima, Shayne V Manarang, April B Lingat, Mariel Niña M
Esguerra, Billy K Singian
Our Lady of Fatima University, Philippines

Abstract
Diplazium esculentum or “Pako” is an edible fern popularly used in homemade salads in the Philippines and the other form is used
as an ornamental plant. According to the findings of Chawla et al. (2015) D. esculentum is a potential analgesic drug in treating
pain and the active constituents inferred to be responsible for the effect were flavonoids and sterols. The present study was done to
evaluate the analgesic property of the semi-purified flavonoids of D. esculentum ethanolic extract. Phytochemical analysis shown
the presence of saponin glycosides, flavonoids, proteins and fixed oils. The evaluation used acetic acid induced writhing method in
five groups of swiss albino mice given the dose of (25, 100, 300 and 500 mg/kg) of the plant extract compared to the positive
control (Aspirin). The study used One Way Anova and Tukey’s Multiple Comparison test in the analysis of the data. The
computed F which is 153.7977528 is greater than the F-critical which is 2.866081402 therefore there is a significant difference
between the groups. The comparison used 95% level of confidence with a P value of 0.1209. These data may indicate that the
semi-purified flavonoids of D. esculentum has showed analgesic effect as the dose increases.

Keywords: analgesic, Diplazium esculentum, flavonoids, pteridophytes, aspirin

Introduction beneficial uses and the researchers focused on identifying its

Philippines is a tropical country with great biodiversity reason medicinal use.
why many plants were discovered and cultivated until today. According to the recent findings of Chawla et al., 2015 [7]. D.
In ancient times, Filipinos utilize folklore herbal medicines in esculentum is a potential analgesic drug in the treatment of
treating various illnesses such as pain. The availability of pain and further studies should be conducted to develop the
plants is not a problem to consider, the nature itself of the property of this plant. It concluded that the active constituents
country supports the needs of its people in searching for responsible for the protective action were the flavonoids and
unconventional medicines. However because of the changing sterols. Therefore, the researchers of this study decided to
society, people ascertained different technologies and ways on continue the determination of the constituent that elicit the
how to obtain drugs in a convenient way. Nowadays, several desirable effect and flavonoids from the ethanoic extract of D.
drugs of chemical source emerged in the market and one of esculentum was subjected for evaluation of its analgesic
these are the analgesics. Large number of the population property.
prefers chemically obtained drugs rather than the natural or
plant sources in the remedy of pain. Background of the Study
Pain is a tormenting unpleasant sensation that can lead from Several drugs for pain such as low-back pain, headache,
general to localized discomfort. The sensation is characterized arthritis pain, cancer pain, and other conditions have several
as both physical and emotional. Patients can now select their limitations including restricted long-term efficacy, tolerance
treatment from a wide range of analgesic drugs in the market problems and other adverse effects. Therefore, herbal
and some are available over the counter or those which are medicine is the answer for a natural way of treating pain and
dispensed without a prescription. Aside from chemical origin, Pteridophytes is an example of a plant with various uses in
plants are also a basis of analgesic properties in which the food, medicine and other necessities in life.
researchers are interested to. In this study, the researchers used Diplazium esculentum also
“Pako” or Diplazium esculentum is an edible fern popularly known as “Pako” in the Philippines. The fresh young ferns of
used in homemade salads in the Philippines and the other form this plant is commonly served as a salad which is rich in
is used as an ornamental plant. It belongs to the family vitamins and other nutrients while the matured plant is usually
Athyriaceae and widely distributed in Asia and to other parts used as a decoration in flower arrangements just like the other
of France, North America, Hawaii and Polynesia. The plant types of ferns in the same family. Studies regarding this plant
usually originates on stony bars and near banks of streams. are emerging with different findings and conclusions such as
The stipe grows 20-50 centimeters long and green in color its antioxidant, antimicrobial, cytotoxic and analgesic activity.
(Quisumbing). They are rich in calcium, phosphorus and a Therefore, the researchers of this study were focused in the
good source of iron and vitamin B. This plant has a lot of evaluation of its property to lessen the different types of pain

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in the body without producing such tolerance problems and last 2014. Accordingly, the dried rhizomes of the plant are
adverse effects like those drugs commercially available in the mainly used as insecticides and it is also used for coughs and
market. haemoptysis through the method decoction. In the folklore
medicine, tribal and ethnic groups utilized the various parts of
Theoretical Framework the plant in the treatment of various ailments. The leaves are
Diplazium esculentum is one of the most common used for headache, pain, fever and wounds. The plant is also
pteridophytes and distributed widely in Asia. According to used in dysentery, glandular swellings, indigestion, diarrhea
Quisumbing, trunk bears many black wiry roots; it is erect, and various skin infections. Phytochemical consttuents are
woody and thick. The stalks are green in color and somewhat steroids, triterpenoids, phenols, flavones, flavonoids such as
smooth; it is also 20-50 cm long. The tip is clothed with myrcetin and alphatocopherol.
brown linear scales. Fronds are 2 or 3 pinnate, 50-80 cm long, For the determination of the total flavonoid content, Folin-
and about half as wide as long. The pinnate are broad ovate, 2- Ciocalteu reagent was used. The methanol extract revealed the
3 cm long and rather coarsely toothed. The clusters of spore presence of various plant secondary constituents while the
are located on the side of the veins or veinlets. chloroform extract yielded no result for carbohydrates.
The recent findings of Chawla et al, stated that the plant was Flavonoids are polyphenols commonly found in fruits,
found to be a potent analgesic and flavonoids and sterols were vegetables and plants. Flavonoids contribute significantly as
the attributes that may be considered in eliciting the protective natural antioxidant. Flavonoids have been shown to possess
action. The intention of the researchers is to evaluate the antimutagenic and antimalignant effect along with its role in
flavonoids of D. esculentum using ethanolic extract and in its preventing diseases like cancer and inhibiting low density
semi-purified form. Flavonoids are polyphenols commonly lipoprotein (LDL) oxidation induced by free radicals.
found in fruits, vegetables and plants. This constituent is Flavonoids have also shown activity as antibacterial, antiviral,
significantly used as natural antioxidant and has been shown antitumor and anti-inflammatory.
to possess antimutagenic and antimalignant effects. The The study concluded that D. esculentum leaf extracts possess
solvent ethanol has the ability to extract more phytochemical antimicrobial activity and cytotoxic activity. The antioxidant
constituents such as phenolics and flavonoids. potential of the plant was exhibited by the reducing capacity
In the evaluation of the analgesic property acetic acid induced of flavonoids and cupric ion.
through the process of writhing method was utilized and
aspirin served as the positive control. The method is usually Local study
applied in peripherally acting analgesics and aspirin is a non- A local study of Amoroso et al published last January 2014,
opioid that has analgesic property that acts by suppressing from Central Mindanao University, entitled “Antimicrobial,
pain substances in the peripheral tissue. Furthermore, pain Antipyretic, and Anti-Inflammatory Activities of Selected
perception involves the production of prostaglandin and Philippine Medicinal Pteridophytes” provided the researchers
flavonoids were reported to inhibit prostaglandin synthetase. the necessary information in conjugation to the phytochemical
screening of the plant Diplazium esculentum. According to the
Literature Review study, Pteridophytes are described to as herbal medicines used
This chapter provides information regarding literature and in the treatment of various illnesses yet scarce literature
studies conducted about Diplazium esculentum. Further, it regarding the medicinal potentials of this plant have been
presents the foreign and local literature and foreign and local conducted. The study used 15 species of Philippine medicinal
studies. pteridophytes in the determination of the constituents of each
plant specifically alkaloids, flavonoids, saponins, tannins and
Local Literature anthraquinones. The antimicrobial activity of the plants were
“Fiddlehead fern” also known as “Pako” is from the family tested on Candida albicans, Staphylococcus aureus,
Atheriaceae which is abundant in the southern parts of the Escherichia coli, Bacillus subtilis, and Pseudomonas
island of Luzon up to the entire Central Visayas. This study Aeruginosa. Egg albumin induced paw edema was the method
was entitled “Nutritional and Phytochemical Screening, and used in the anti-inflammatory activity and yeast induced
Total Phenolic and Flavonoid Content of Diplazium pyrexia for antipyretic activity.
esculentum (Retz.) Sw. from Philippines” by Jovale Vincent The ethanol extraction used 200 g of the fresh plant were
V. Tongco et al. Publish last 2014. weighed separately and 95% ethyl alcohol was used and the
The study concluded that methanol is more toxic and the plant was completely submerged for 24 hours, filtered and
solvent used in the study was ethanol. For its ability to concentrated using a rotary evaporator. The results of the
respond to specific bioassays and its ability to extract more phytochemical screening showed that D. esculentum has
constituents like phenolic compounds and flavonoids rather flavonoids. The anti-inflammatory and antipyretic activities of
than water. the plants are coined in the presence of flavonoids which is an
important pharmacological constituent. Based on the table of
Foreign Literature phytochemical analysis of the ethanolic extract, D. esculentum
Diplazium esculentum is one of the most common is negative in the test for leucoanthocyanins (Bate-Smith and
pteridophytes and distributed widely in Bangladesh and Metcalf). Anthraquinones were undetected in all species of
Philippines. This study was entitled, “Investigation of In vitro Pteridophytes used in the study. The absence of
Antioxidant, Antimicrobial and Cytotoxic activity of anthraquinones in the pteridophyte extract may prevent the
Diplazium esculentum (RETZ). SW.” by Akter et al published occurrence of diarrhea when the plant extracts are

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administered orally as herbal remedy. The study used laboratory animals in the evaluation of the
Extract form three species of pteridophytes namely: analgesic property of D. esculentum and the 30 Swiss albino
Equisetum ramosissimum, Pyrrosia piloselloides, and mice were purchased from the Pharmacology and Toxicology
Selaginella usterii did not manifest any antibacterial activity Department of University of the Philippines, Manila. The
and all extracts showed significant effect in reducing yeast- researchers submitted documents for Application of
induced pyrexia when compared with the negative control Authorization and Animal Care and Use Statement (IACUC)
(NSS). at Our Lady of Fatima University. The study also passed the
OLFU Institutional Ethics Review Committee (IERC).
Foreign Study The fresh plant samples were collected near banks of streams
The plant Diplazium esculentum was recently studied by at Gloria 1 Sindalan City of San Fernando, Pampanga on the
Chawla et al. in the year 2015 from the Department of month of August. For the certification of authenticity, the
Pharmacy, Himachal Institute of Pharmacy, India entitled plant materials were brought to the Botany Division of the
“Analgesic Activity of Medicinally Important Leaf of National Museum, Manila.
Diplazium esculentum”. The prospect of the study entails the The 100 grams of the plant materials were air dried for seven
use of medicinal plants in modern era in the cure of pain. days. The dried fronds were reduced in particle size, weighed
Fresh plants were gathered and were air dried at temperatures and placed in a wide mouth glass container. The pulverized
ranging from 25oC to 35oC, reduced, and preserved. The plant samples were extracted using the solvent 80% ethyl
researchers used four solvents (Extract I-distilled water, alcohol and macerated for 72 hours with frequent agitation.
Extract II-petroleum ether, Extract III-chloroform, and Extract The extracted fronds of Diplazium esculentum were screened
IV-ethanol) in extracting the plant constituents and the for the presence or the absence of flavonoids, alkaloids,
phytochemical screening yielded flavones, flavonoids, glycosides, proteins, carbohydrates, saponins, tannins, and
proteins, steroids, and triterpenoids respectively. phenolic compounds.
Acetic acid induced the practice of writhing process. It was To obtain the semi-purified flavonoids, the collected plant
utilized for the determination of the analgesic activity. Six (6) samples were air dried for 7 days and the 100 grams of the
groups of albino mice of both genders ranging from 20 to 30 plant was submerged and macerated with 80% ethyl alcohol
grams in weight were grouped into two and each group for 48 hours. Using a Rotary Evaporator, the crude extract was
includes 5 mice. The first group serves as the control and the concentrated until a viscous solution was obtained. This was
second group was treated with the standard drug (aspirin). evaporated to dryness in a water bath at 60 degrees Celsius.
Route of administration was intraperitoneally at a dose of The residue was treated with almost 10 mL of 2M HCl and
25mg/kg and the biological animals were placed and observed equal amounts of ethyl acetate. The treated extract was
under a glass jar. The response such as trunk twists, extension evaporated to incipient dryness and the residue left was the
of the hind limbs and abdominal contractions were regarded as flavonoids. The presence of the constituent was confirmed
the writing responses. It was calculated that the reactions using alkaline reagent test.
produced by the extract commenced after 10 minutes of The semi-purified flavonoids were subjected to organoleptic
administration. Based on the level of significance the results evaluation, solubility, confirmatory tests and acute oral
of the acetic acid induced writhing method states that extract toxicity tests.
IV or the ethanol solvent is greater than the results of extracts Acute oral toxicity test was performed following the
I, II, and III. Organization for Economic Cooperation and Development
The study concluded that the aqueous extracts of Diplazium (OECD) guideline # 423. The test animals that were used in
esculentum were found to be potent analgesic. Based on the the study were nulliparous, non-pregnant, and healthy female
observations it has a marked advantageous effects against albino swiss mice.
centrally, peripherally and inflammatory pain models. The test animals were kept and placed in an experimental
Flavonoid and sterols were the attributes that may be room with temperatures ranging from 22oC (+3oc). Animals
considered in eliciting the protective action. Accordingly, the were given unlimited supply of water and will be grouped. For
study encourages further studies on the matter to identify the the preparation, the test animals were randomly selected,
exact active constituent accountable for the analgesic activity given identification and were placed in the cage 5 days prior
of the plant. There reports may serve as a footstep in the to dosing. The samples were administered in a constant
research of a potent analgesic drug. (Chawla et al, 2015) [7]. volume according to the range of doses per concentration.
Prior to dosing, the mice were fasted for 3-4 hours.
Research Methodology Administration of the test substance was of single dosing and
The study was conducted to evaluate the analgesic property of was administered via oral gavage using a suitable intubation
Diplazium esculentum fronds. Randomized control trials were cannula. After the administration of the test substance the
used in the manipulation of the quantitative and expected mouse was withheld from food for 1-2 hours. The doses that
results. Experimental method was used by the researchers to were administered were 5, 50, 300 and 2000 mg/kg body
be a tool in the evaluation of the analgesic property of D. weight and were observed for 30 minutes, 24 hours until 14
esculentum. The use of experiments in the course of the study days.
was to predict and to answer the stated problems by For the determination of the analgesic property of D.
manipulating the given variables. The method was most esculentum, six groups of swiss albino mice weighing 20
applicable in every aspect of the study from the extraction of grams of the female gender containing 5 in each group was
plant constituents to the isolation and biological tests. used in the study. Four groups were for the plant extract with

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different dose (25, 100, 300 and 1000mg/kg), one group for The identification of the functional groups of Diplazium
the negative control, and one group for the standard drug esculentum was conducted using the Fourier Transform
which was aspirin (positive control). The method used was Infrared Spectrophotometry. The interpretation of results in
acetic acid induced writing method. All the extracts and the table 2 was coined from the infrared absorbance spectrum of
standard drug were dissolved in distilled water. The negative the chemical bonds. The wavelength of light absorbed that
control (NSS), were administered via oral gavage 1ml/100g of was seen in the annotated spectrum is hereby recorded. The
normal saline. At a dose of 25 mg/kg, the standard drug functional groups of D. esculentum were O-H (Alcohol
(aspirin) was administered orally as well as the 25, 100, 300, Phenol), C-H (Alkyl), C=O (Amide), Nitro compounds and C-
and 1000 mg/kg of the semi-purified flavonoids. After 15 O (Alkoxy) groups. The analysis of results was according to
minutes, 3% of acetic acid solution (1ml/100g) was Bruker alpha FTIR Spectrometer.
administered intraperitoneally. The animals were individually
observed in a glass jar. The trunk twist response, extension of Table 3: Results of the Organoleptic Evaluation of The Semi-
limbs and abdominal contractions were recorded for 10 purified Flavonoids of D. esculentum Ethanolic Extract
minutes. Test Result
Appearance Viscous
Results and Discussions Color Yellow
In this chapter, data is presented, analyzed and interpreted. Odor Pungent
Presentation and analysis were accomplished through tables
and narratives. Data were interpreted in light of information The table 3 represents the result of the organoleptic test of the
gathered during the review of relevant theories, literature and Semi-purified Flavonoids of Diplazium esculentum ethanolic
studies. extract.
The semi-purified flavonoids appeared to be viscous, yellow
Table 1: Results of the Phytochemical Screening of the D. in color with pungent odor. The alkaline reagent test for the
esculentum Ethanolic Extract confirmation of flavonoids produced the formation of intense
yellow color, which disappeared upon addition of diluted acid.
Phytoconstituents Test Remarks
Froth Test -
Saponin Glycosides Table 4: Results of the Solubility Test of The Semi-purified
Liebermann-burchard’s Test ++
Cardenolides and Flavonoids of D. esculentum Ethanolic Extract
Keller Kelliani Test -
Bufanolides Solvent Result
Leucoanthocyanins (bate-smith Water Soluble
-
Flavonoids and metcalf’s test)
Petroleum Ether Insoluble
Willstatter cyanidin test ++
Methanol Soluble
Borntrager’s Test -
Anthraquinone Acetone Soluble
Modified Borntrager’s Test -
Wagner’s Test -
Chloroform Insoluble
Alkaloids
Mayer’s Test -
Total Phenolic Gelatin’s Test - The table 4 represents the result of the solubility test of the
Compounds Ferric chloride Test - Semi-purified Flavonoids of Ethanolic Extract from the
Fehling’s Test A and B - Diplazium esculentum Fronds. Using the five solvents for the
Carbohydrates
Moore’s Test - solubility test, the semi-purified flavonoids were soluble in
Xanthoproteic Test ++ water, acetone and methanol yet insoluble in chloroform and
Proteins
Millon’s Test -
petroleum ether. (Tiwari et al, 2011)
Fixed Oils and
Stain Test Fixed Oil
Volatile Oils
The table 5 shows the results of the 14 days of observation for
The table 1 presents the result of the phytochemical screening the potential toxicity of the semi-purified flavonoids according
of the ethanolic extract of D. esculentum. Based on the test to general. There were no signs of toxicity and mortality
performed, the group obtained positive result on saponin among the test animals throughout the entire observations for
glycosides, flavonoids, proteins and fixed oils. This means the acute toxicity studies. All the mice exhibited normal
that the constituents are present on the sample. behavior, usual movement patterns and were all in healthy
According to Tongco et al, ethanol is desired in the analysis of condition after the administration of the doses (5, 50, 300 and
the phytochemical constituents due to its ability to produce 2000 mg/kg). The examination lasted for 14 days and the
more constituents than water alone. largest dose produced no signs of mortality. Therefore, the
ethanolic extract of the semi-purified flavonoids of Diplazium
Table 2: Interpretation of the FTIR Spectrum of the Semi-purified esculentum is safe to be used as a medicinal plant with no
Flavonoids of D. esculentum Ethanolic Extract toxicity. The median lethal dose (LD50) of this plant was
Wave Numbers (Cm-1) Types Of Vibrations Functional Group
estimated to be above 2000 mg/kg body weight. (Junejo et al,
3263.92 OH Stretch Alcohol-Phenol 2015)
2944.96 C-H Stretch Alkyl
1637.16 C=O Stretch Amide
1052.58 C-O Stretch Alkoxy

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Table 5: General behavioral observations for acute toxicity studies of the semi-purified flavonoids of D. esculentum ethanolic extract
Observation Time Trial no. 1 Trial no. 2 Trial no. 3 Trial no. 4 Trial no. 5
30 minutes Normal Normal Normal Normal Normal
Tremor 24 hours Normal Normal Normal Normal Normal
14 days Normal Normal Normal Normal Normal
30 minutes Normal Normal Normal Normal Normal
Eyes and Skin 24 hours Normal Normal Normal Normal Normal
14 days Normal Normal Normal Normal Normal
30 minutes Normal Normal Normal Normal Normal
Behavioral Patterns 24 hours Normal Normal Normal Normal Normal
14 days Normal Normal Normal Normal Normal
30 minutes Normal Normal Normal Normal Normal
Lethargy and Sleep 24 hours Normal Normal Normal Normal Normal
14 days Normal Normal Normal Normal Normal
30 minutes Normal Normal Normal Normal Normal
Allergic Reaction 24 hours Normal Normal Normal Normal Normal
14 days Normal Normal Normal Normal Normal
30 minutes Normal Normal Normal Normal Normal
Mortality and Coma 24 hours Normal Normal Normal Normal Normal
14 days Normal Normal Normal Normal Normal

Table 6: Number of Writhes of Groups A, B, C, D, E and F

Number of Writhes
GROUP # Dose Administered
Trial no. 1 Trial no. 2 Trial no. 3 Trial no. 4 Trial no. 5 Mean Percentage inhibition
A (Negative Control) 1 mL/100g 38 35 33 36 35 35.5 Xx
B (Aspirin) 25 mg/kg 15 12 14 16 13 14 60.45%
C (Experimental Control) 25 mg/kg 32 30 29 28 31 30 15.25%
D (Experimental Control) 100 mg/kg 22 24 23 22 21 22.4 36.72%
E (Experimental Control) 300 mg/kg 16 16 17 16 17 16.4 53.67%
F (Experimental Control) 1000 mg/kg 14 10 11 12 11 11.6 67.23%

The table 6 represents the raw data of the number of writhes to be the dose wherein the plant exhibited its analgesic effect.
recorded from the 5 groups of test animals. The data shows the Based on the raw data of the number of writhings, it was
decrease in writhing responses as the dose increases. showed that as the dose increases, the number of writhing
The analgesic property was computed using percent inhibition response decreases. This confirms the conclusion of (Chawla et
and this was implied by the decrease in writhing responses. In al, 2015) [7] in the study of the medicinal property of D.
accordance to this, Group F which was the experimental control esculentum which is the main objective of this study.
produced the highest mean of 67.23% and this was considered

Table 7: One Way Anova Interpretation of Results

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 1095.04 4 273.76 153.7977528 1.02338E-14 2.866081402
Within Groups 35.6 20 1.78
Total 1130.64 24

The table 7 represents the result of the One Way Anova and difference between the groups.
the interpretation stated that the null hypothesis is rejected The Tukey’s Multiple Comparison Test used 95% level of
and the alternative hypothesis was accepted. confidence in the comparison of the individual groups. Group
The study used one way anova and Tukey’s multiple E and Group F versus the positive control resulted in adjusted
comparison test in the analysis of the data. The statistical P value of 0.1209 indicating that among the 5 groups with a P
treatment between the analgesic property of the positive value of <0.0001, Group E and Group F has significant
control (Aspirin 25 mg/kg) and the experimental control (D. analgesic property.
esculentum semi-purified flavonoids 25, 100, 300, and This data may indicate that the semi-purified flavonoids of D.
1000mg/kg) showed that the computed F which is esculentum has showed analgesic effect as the dose increases.
153.7977528 is greater than the F-critical which is (Chawla et al, 2015) [7].
2.866081402 in anova, therefore there is a significant

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Table 8: Tukey’s Multiple Comparison Test Interpretation of 3. Further evaluation of other plant parts of Diplazium
Results esculentum as source of flavonoids other than its fronds.
Tukey's multiple Adjusted 4. To conduct further studies on the semi-purified flavonoids
Mean Diff. Summary
comparisons test P Value of D. esculentum such as formulating it into a dosage
A vs. B 21.4 **** <0.0001 form.
A vs. C 5.4 **** <0.0001
5. To conduct other methods of determination of the
A vs. D 13 **** <0.0001
A vs. E 19 **** <0.0001
analgesic property of D. esculentum.
A vs. F 23.8 **** <0.0001
B vs. C -16 **** <0.0001 References
B vs. D -8.4 **** <0.0001 1. Jovale Vincent V Tongco, Ronald Arlet P Villaber, Remil
B vs. E -2.4 Ns 0.1209 M Aguda, Ramon A Razal. Nutritional and phytochemical
B vs. F 2.4 Ns 0.1209 screening, and total phenolic and flavonoid content of
C vs. D 7.6 **** <0.0001 Diplazium esculentum Retz. Sw. from Philippines. 2014;
C vs. E 13.6 **** <0.0001 6(8):238-242
C vs. F 18.4 **** <0.0001 2. Julfikar Ali J, Anirban G, Prodyut M, Lalit N, Kamaruz Z,
D vs. E 6 **** <0.0001 Khumanthem DS et al. In-vivo Toxicity Evaluation and
D vs. F 10.8 **** <0.0001 Phytochemical, Physicochemical Analysis of Diplazium
E vs. F 4.8 *** 0.0002 esculentum Retz. Sw. leaves a traditionally used North-
The table 8 represents the results of Tukey’s Multiple Comparison Eastern Indian Vegetable. Adv. Biores. 2015; 6(5):175-
Test using 95% level of confidence. 181. DOI:10.15515/abr.0976 4585.6.5.175181
3. Muhammad Razi Ullah Khan, Saeed Ur Rashid Nazir,
Conclusion Musaddique Hussain. Formulation Design, Optimization
The conclusions were drawn based on the observation and and Anti- Inflammatory Evaluation of Ibuprofen Cream.
findings of the study made on the Diplazium esculentum 2014; 4(5):762-763.
fronds. The semi-purified flavonoids of ethanolic extract of 4. Nitin Kharat, Shylaja H, Viswanatha GL, Lakshman K.
D. esculentum yielded positive result in the Wilstatter Anti- Inflammatory and Analgesic Activity of Topical
“Cyanidin” Test. It also possess the characteristic features of Preparation of Root Extracts of Ichnocarpus frutescens.
the flavonoids proven by the result from the physical, 2010; 1(3):1103.
chemical, and instrumental test for assay conducted. The 5. Prashant Tiwari, BimleshKumar, Mandeep Kaur,
semi-purified flavonoids of the ethanolic extract of D. Gurpreet. Phytochemical screening and Extraction: A
esculentum showed no signs of toxicity and mortality in acute Review. 2011; 1(1):98-106.
toxicity studies. There is also a reduction in the number of 6. Ashokkumar R, Ramaswamy M. Phytochemical screening
writhings of the experimental control (Group E 300 mg/kg by FTIR spectroscopic analysis of leaf extracts of selected
and F 1000 mg/kg) which may indicate that the semi-purified Indian Medicinal plants. 2014; 3(1):395-406.
flavonoids of D. esculentum has analgesic property as stated 7. Sonia Chawla, Saurabh Chawla, Veerma Ram, Alok
on the recent findings of Chawla et al, 2015) [7]. The statistical Semwal, Ramandeep Singh. Analgesic activity of
treatment showed that there is a significant difference medicinally important leaf of Diplazium esculentum
between the positive and experimental control and showed Dehradun-248161, U.K. India, 2015; 9(25):628-63.
that the computed F value which is 153.7977528 is greater 8. Hajare SW, Suresh Chandra, Tandan SK, Sarma K, Jlal.
than the F-critical which is 2.866081402 in anova, therefore Analgesic and Antipyretic Activities of Dalbergia Sisoo
there is a significant difference between the groups. This data Leaves. Indian Journal of Pharmacology, 2000, 1-2.
may indicate that the semi-purified flavonoids of D. 9. Victor B Amoroso, Dorothy A Antesa, Dave P
esculentum have showed analgesic effect as the dose Buenavista, Fulgent P Coritico. Antimicrobial,
increases. It resulted in adjusted P value of 0.1209 indicating Antipyretic, and Anti-Inflammatory Activities of Selected
that among the 5 groups with a P value of <0.0001, Group E Philippine Medicinal Pteridophytes, 2014; 5:18-40.
and Group F has significant analgesic property. 10. Julfikar Ali Junejo, Anirban Ghoshal, Prodyut Mondal,
In accordance to the study of Chawla et al. entitled Lalit Nainwal, Kamaruz Zaman, Khumanthem Deepak
“Analgesic activity of medicinally important leaf of Singh, Tapash Chakraborty. In-vivo Toxicity Evaluation
Diplazium esculentum” the results have showed that the and Phytochemical, Physicochemical Analysis of
protective action was attributed between flavonoids and Diplazium esculentum Retz. Sw. leaves a traditionally
sterols. Therefore the researchers of this study concluded that used North-Eastern Indian Vegetable.
flavonoids is responsible for the Analgesic property. 11. Sujith S. Nair, Molly Mathew, Sreena K. Formulation and
Evaluation of Herbal Cream, 2012.
Recommendations
The Researchers would like to recommend the following:
1. Further evaluation of the other uses of the semi-purified
flavonoids of Diplazium esculentum fronds.
2. Further evaluation of other constituents like sterols
present in Diplazium esculentum that potentially elicits
Analgesic property.

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