Skip to main content

JPRDP 7

% DPPH scavenging activity at 0.5 mg/ml

                       Njinga et al., J. Pharm. Res. Dev. & Pract., December, 2016, Vol. 1 No. 1, P 69-78 ISSN:2579-0455

 

Comparative phytochemical analysis and antioxidant activity of the stem and seed of telfairia occidentalis hook. F. (cucurbitaceae)

N.S.NJINGA*1, M.T.  BAKARE-ODUNOLA1, S.T ABDULLAHI1, J.A OKONKWO2, O.H  OLADIMEJI3

        1. Department of Pharmaceutical and Medicinal Chemistry, University of Ilorin, Ilorin, Nigeria.

             2.Faculty of Pharmaceutical Sciences, University of Ilorin, Ilorin, Nigeria.

3. Department of Pharmaceutical and Medicinal Chemistry, University of Uyo, Uyo, Nigeria.

_______________________________________________________________________

 *Corresponding Author’s E-mail: ngastanjin@yahoo.com. GSM: +2347064933604

ABSTRACT

This research analyzed and compares the phytochemicals and antioxidant activity of the different fractions (petroleum ether, chloroform, ethyl acetate, acetone and methanol) of the seed and stem extracts of T. occidentalis. Phytochemical studies were done using standard methods and spectrophotometric techniques. The antioxidant activities were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effects. The phytochemistry of the seed fractions showed the presence of cardiac glycosides, alkaloids, steroids and terpenoids while that of the stem extract showed the presence of reducing sugars, alkaloids, cardiac glycosides, flavonoids, steroids and terpenoids. Only the chloroform and ethyl acetate fractions of the stem contain flavonoid and phenols while the other seed did not contain any flavonoid and phenols. The petroleum ether and chloroform fractions of the seed contain more alkaloid than the stem. The difference was statistically significant at p<0.05. Also, only the ethyl acetate and acetone fractions of the stem contains alkaloid while those of the seed did not. The antioxidant activity of the seed fractions (0.5 mg) was found to range from 40.5 to 76.75 % scavenging activity for all the fractions while only the chloroform and ethyl acetate of the stem showed scavenging activity of 69.27 and 76.75 % respectively. The phytochemicals have been reported to be responsible for the pharmacological effect of most medicinal plants. Compounds from the chloroform and ethyl acetate fractions of the stem may be potent leads for potential antioxidant.

_______________________________________________________________________

Key words: Telfairia occidentalis, phytochemical, antioxidant, cucurbitaceae

Introduction

Poverty and limited access to modern medicine has caused about 80% of the world’s population, especially in the developing countries to use herbal medicine as their source of primary healthcare 1,2.

Medicinal plants are widely distributed throughout the world but most abundantly in tropical countries. It is estimated that about 25% of all modern medicines are directly or indirectly derived from higher plants 3. The use of herbs as medicine is the oldest form of healthcare known to humanity and has been used in all cultures throughout history 4.

In many communities, traditional medical practice is often viewed as an integral part of their culture. Herbal medicines are often viewed as a balanced and moderate approach to healing. In the West, like our ancestors, it is believed they will help us live healthier lives. Individuals who use them as home remedies and over-the-counter drugs spend billions of dollars on herbal products. As such, they represent a substantial proportion of the global drug market 3,5.

Although it is generally believed that most herbal preparations are safe for consumption, some herbs like most biologically active substances could be toxic with undesirable side effects 6.  A well-defined and constant composition of the drug is therefore, one of the most important prerequisites for the production of a quality drug. Given the nature of products of plant origin, which are not usually constant and are dependent on or influenced by many factors, ensuring consistent quality of products is vital for the survival and success of the pharmaceutical industry 7.   

Telfairia occidentalis commonly called fluted pumpkin occurs in the forest zone of West and Central Africa, most frequently in Benin, Nigeria and Cameroon 8. The plant is dioecious, perennial, and drought-tolerant. It is a creeping vegetable shrub that spreads low across the ground with large lobed leaves, and long twisting tendrils. Herbal preparations of the plant has been employed in the treatment of anaemia, GIT disorders, malaria, convulsion, impaired defense immune systems and diabetes 9,10,11. The plant has been reported to contain flavonoids, tannins, reducing sugars, glycosides, saponins, steroids and triterperoids 12. This plant has also been reported to have antioxidant, antidiabetic, anti-inflammatory, hepatoprotective, haematinic, properties13,14,15. GC-MS analysis of hexane and dichloromethane fractions of the seed showed that the seed contained compounds such as pentadecanoic acid, hexadecanoic acid; 16- octadecenoic acid methyl ester; 9, 12- octadecadienoyl chloride (Z,Z); 9- Octadecadienoic acid (Z)-, 2, 3-dihydroxypropyl ester; Octadecanoic acid ; hexadecanoic acid, 2,3 bis [(trimethyl-silyl) oxy] propyl ester in the hexane fraction and 2,4-heptadien-6-ynal, (E,E); benzoic acid ; dodecanoic acid ; linoleic acid ethyl ester ; hexadecanoic acid, methyl ester ; α-phellandrene ; α-campholene aldehyde;terpinen- 4-ol ; trans-β-ocimene ; borneol ; stigmastan-3-ol, in the dichloremethane fraction 11.

The research aimed to analyse the phytochemicals and compare the antioxidant activities of the petroleum ether, chloroform, ethyl acetate, acetone and methanol fractions/extract of the seed and stem of Telfairia occidentalis.

 

Materials and Methods

Materials and Equipments

The materials and chemicals used in this study were all BDH products and they include: chloroform, ethylacetate, acetone, methanol, aluminium chloride, sulphuric acid, hydrochloric acid, ferric chloride, glacial acetic acid, acetic anhydride. Other reagents used include: Fehling’s solutions A and B, Wagners’ reagent which were all of analytical grade obtained from the Department of Pharmaceutical and Medicinal Chemistry.

The equipment used include: OHAUS analytical balance, electric oven, water bath, rotary evaporator (SHZ-III, Brinkmann Instruments, Inc., Westbury, New York), UV-Vis spectrophotometer (752N), Milling machine (Viking Type YL8024).

 

Methods

Collection and identification of plant

Fresh shrub and pumpkin of Telfairia  occidentalis were obtained from Olunlade and Pipeline areas, Ilorin, Kwara state, Nigeria. A Taxonomist in the Department of Plant Biology, University of Ilorin, Nigeria, identified the plant as Telfairia occidentalis with voucher number: UILH/001/1063.

 

Preparation and Extraction

The stem and seeds were cut differently into smaller pieces and air dried at room temperature for a week. It was further dried in the oven at 40 0C. The dried plant part was then pounded using a mortar and pestle and then milled in a milling machine.

The milled stem (232g) and seed (300g) of T. occidentalis each were weighed, placed in different extraction bottles and 600 ml each of petroleum ether was poured into it followed by stirring at two hour interval for up to 12 hours after which it was decanted. This process was repeated for 3 days for exhaustive extraction. The decanted supernatant was filtered and the filtrate concentrated using a rotary evaporator. The marc was dried and sequential extracted with chloroform, ethyl acetate, acetone and methanol respectively, to obtain different fractions/extract of the stem and seed of T. occidentalis.

The percentage yield for each fraction of the extracts was calculated using the expression

 

% yield=Weight of extractWeight of powdered plant part x1001

Qualitative phytochemistry

The different fractions of the stem and seed of T. occidentalis were tested for the presence of phytochemicals using the following standard methods 16,17.

 

Test for carbohydrates

Fehling’s test

Equal volumes of Fehling’s solution A and B reagents were mixed together and 2ml of the resulting solution was added to each of the extract and gently boiled. A brick red precipitate appearing at the bottom of the test tube indicated the presence of reducing sugars.

 

Molisch’s test

Each extract was mixed with 2ml of Molisch’s reagent and the mixture was shaken properly. After that, 2ml of concentrated sulphuric acid (H2SO4) was poured carefully along the side of the test tube. Appearance of a violet ring at the interphase indicated the presence of carbohydrate.

 

Test for phenols and tannins

Each extract was mixed with 2ml of a 2% solution of FeCl3. A blue-green or black coloration indicated the presence of phenols and tannins.

 

Test for flavonoids

Each extract was mixed with 2ml of a 2% solution of NaOH. The formation of an intense yellow colour which turned colourless on addition of few drops of diluted HCl indicated the presence of flavonoids.

 

Test for saponins

Each extract was mixed with 5ml of distilled water in a test tube and it was shaken vigorously. The formation of a stable foam was taken as an indication for the presence of saponins.

 

Keller-kilani test for glycosides

Each extract was mixed with 2ml of glacial acetic acid containing 1-2 drops of a 2% solution of FeCl3. The mixture was then poured into another test tube containing 2ml of concentrated H2SO4. A brown ring at the interphase indicated the presence of cardiac glycosides.

 

Test for steroids

Each extract was mixed with 2ml of chloroform and concentrated H2SO4 was added sidewise. A red colour produced in the lower chloroform layer indicated the presence of steroids. Another test was performed by mixing the crude extract with 2ml of chloroform, and then 2ml of each of concentrated H2SO4 and acetic anhydride were poured into the mixture. The development of a greenish colour indicated the presence of steroids.

 

Test for terpenoids

Each extract was dissolved in 2ml of chloroform and 2ml of concentrated H2SO4 was carefully added. A layer of reddish-brown colour at the interphase indicated the presence of terpenoids.

 

Test for alkaloids

Each extract was mixed with 2ml of 1% HCl and heated gently. Wagner’s reagent was then added to the mixture. Turbidity of the resulting precipitate was taken as evidence for the presence of alkaloids.

 

Quantitative phytochemistry

Total Phenolic content

The amount of phenol in the extract was determined by the Folin-Ciocalteu reagent method 18 with some modifications. 3.6 ml of 10 % Folin-Ciocalteu reagent and 2.9 ml of 7 % solution of Na2CO3 were added to 3.6 ml of the plant extract. The resulting mixture was incubated for 30 minutes at room temperature and the absorbance of the solution was measured at 765 nm. All extracts were evaluated at a final concentration of 0.1 mg/ml. Total phenolic content was expressed as mg/g tannic acid equivalent using the following equation based on the calibration curve: y = 0.1216x, R2 =0.9365, where y was the absorbance x was the concentration.

 

Total Flavonoid content

The aluminium chloride colorimetric method 19 was used with some modifications to determine the flavonoid content. 0.5 ml of sample plant extract was mixed with 2 ml of methanol, 2.5 ml of 10 % aluminium chloride and kept at room temperature for 30 minutes. The absorbance was then measured at 420 nm. Extracts were evaluated at a final concentration of 0.1 mg/ml. Total flavonoid content were calculated as quercetin equivalents (mg/g) using the following equation based on the calibration curve: y = 0.0255x, R2 =0.9812, where y was the absorbance x was the concentration.

 

Total Alkaloid determination

0.1 g of the each extract was weighed into a sample bottle and 4 ml of 20 % acetic acid in ethanol was added. The sample bottle was covered and allowed to stand for 4 h. It was then filtered and the extract was concentrated on a water bath at 55°C to one quarter of its original volume.  Concentrated ammonium hydroxide was added dropwise to the extract until precipitation was complete. The precipitate was collected from the solution and washed with dilute ammonium hydroxide and filtered. The residue, which contained the alkaloid was weighed after dryness to completion and the percentage was calculated 20 using the formular:

% alkaloid  =Weight of alkaloid precipitateWeight of powdered sample x1001

 

Antioxidant Assay

This was done using DPPH 21.  0.5 mg/ml of each extract was added, at an equal volume, to methanolic solution of DPPH (100 µM). The mixture was kept in the dark at room temperature for 30 minutes. The absorbance (A) was then measured at 518 nm and converted into the percentage antioxidant activity using the following equation:

% DPPH   =Absorbance DPPH-Absorbance (Extract)Absorbance (DPPH)x1001

 

Statistical Analysis

All experiments were done in triplicates  and data obtained were represented in tables as Mean ± Standard error of mean (SEM). Analysis of Variance (ANOVA) was used to statistically analyze the data where applicable. T-test was used to compare the mean data obtained for each extract of the seed and stem of T. occidentalis. Differences between samples were taken as being significant at P < 0.05.

Results and discussion

After exhaustive extraction of the different fractions of the seed and stem extracts of T. occidentalis, chloroform gave the highest yield for the seed and stem (7.61 ± 0.05 and 1.07 ±  0.03 % respectively) while ethyl acetate afforded the lowest yield (0.09 ± 0.01%) as shown in table 1. Extractive value in the order: CHCl3 > petroleum ether > ethyl acetate > methanol > acetone and CHCl3> petroleum ether > acetone > methanol > ethyl acetate for the seed and stem fractions respectively (Table 1). This indicated that the phytochemicals extracted are mostly less polar as can be observed from the Table 3 in which the chloroform fraction of the seed contains mostly alkaloids, cardiac glycoside, steroids and terpenoids.

The methanol fraction of the stem is soluble in water because of the presence of flavonoid (Table 2) while that of the seed is soluble in water because of the presence of mostly steroids and cardiac glycoside.

 

 

Table 1. Percentage yield of the seed and stem extract of T. occcidentalis

Plant part

fractions

Weight of powdered drug (g)

Weight of extract (g)

% yield ± SEM

Seed

Petroleum ether

300

14.28

4.76 ± 0.10

Chloroform

300

22.82

7.61 ± 0.05

Ethyl acetate

300

6.17

2.06 ± 0.06

Acetone

300

2.05

0.68 ± 0.03

Methanol

300

3.50

1.17 ± 0.01

Stem

Petroleum ether

232

1.79

0.77 ± 0.03

Chloroform

232

2.49

1.07 ± 0.03

Ethyl acetate

232

0.21

0.09 ± 0.01

Acetone

232

0.60

0.26 ± 0.01

Methanol

232

0.42

0.18 ± 0.01

 

 

The qualitative phytochemistry of Telfairia occidentalis stem (Table 2) showed the presence of reducing sugars and alkaloids in all the fractions except the methanol extract; cardiac glycosides in only the methanol extract; flavonoids in the chloroform and acetone extracts. Steroids were present only in the chloroform extract and terpenoids in only the acetone extract. Saponins and tannins were absent in the extracts of the stem of Telfairia occidentalis. This correlates with previous work where carbohydrates was present 22, and this may imply that the stem of T. occidentalis can be a good source of energy.

Telfairia occidentalis seed showed the presence of cardiac glycosides, steroids and terpenoids in all the extracts and alkaloids in only the petroleum ether and chloroform extracts. Cardiac glycosides were present in high quantity in Telfairia occidentalis seeds. Carbohydrates, reducing sugars, flavonoids, saponins and tannins were absent in all the fractions of Telfairia occidentalis seed extract. This contradicts the work done by Eseyin et al. 22 in which they indicated the presence of carbohydrates and reducing sugars. This could be due to the environmental and climatic factors associated with its plantation, maturity and harvesting.

Secondary metabolites (phenols, flavonoids, alkaloids) are known to have antimicrobial activity 23, Cardiac glycosides use for the treatment of heart failure and atrial arrhythmia 24, are potent inhibitors of DNA double-strand break repair 25, thus suggesting the possible uses for the this plant for medicinal purposes

 

 

Table 2. Qualitative Phytochemistry of the seed and stem of Telfairia occcidentalis

Phytochemical constituent

Test

Petroleum ether

Chloroform

Ethyl acetate

Acetone

Methanol

seed

stem

seed

stem

seed

stem

seed

stem

Seed

stem

Carbohydrates

Molish’s Test

-

-

-

-

-

-

-

-

-

-

 

Reducing sugars

Fehling’s Test

-

+ + +

-

+ + +

-

+ + +

-

+ + +

-

+

Alkaloids

Wagners’ Test

+

+

+

+

-

+ + +

-

+

-

-

Cardiac glycosides

Keller-kilaniTest

+ + +

-

+ +

-

+ +

-

+ + +

-

+

+ +

Flavonoids

Alkaline reagent Test

-

-

-

+

-

-

-

+

-

-

Saponins

Frothing Test

-

-

-

-

-

-

-

-

-

-

Steroids

Acetic anhydride  Conc. H2SO4

+ +

-

+ + +

+ +

+ +

-

+

-

+

-

Tannins

Fecl3Test

-

-

-

-

-

-

-

-

-

-

Terpenoids

CHCl3 + H2SO4

+

-

+

-

+

-

+

+

+

-

                             

KEY:  + = presence;   -  = absence

 

Quantitative Phytochemistry of Telfairia occcidentalis

The result of the total percentage phenolic content showed that the acetone extract contained more phenolic content (4.02%) than the chloroform extract (3.59%) as shown in Figure 1. Phenols have been associated with the antimicrobial activity of the plant 23 thus the presence of phenol in this plant justifies it antimicrobial activity as reported in literature 26

 

 

 

Figure 1. Total phenolic content in the seed and stem of  Telfairia occidentalis. (Values with a superscript are significantly different [P < 0.05].

 

Only the chloroform and acetone extracts of the stem significantly contained flavonoid (31.37 and 3.18%) as shown in Figure 2. Flavonoids are polyphenolic compounds found in a wide variety of plants and they impart a yellow or white pigment to flowers and fruit and have a wide range of activity. Flavonoids are particularly useful for maintaining healthy circulation and some are antioxidant, while others are anti-inflammatory, anti-viral or capillary strengthening. This shows the high medicinal potency of the chloroform and acetone fractions as antioxidants (Figure 4).

 

Figure 2. Total flavonoid content in the seed and stem of Telfairia occidentalis. (Values with a superscript are significantly different [P < 0.05]).

 

The seed of Telfairia occidentalis showed a significantly high (P<0.05) alkaloid content for both petroleum ether and chloroform extracts with total percentage alkaloid content of 40.64% and 29.22% respectively, whereas, the acetone and ethyl acetate extracts of the stem only contained alkaloid (Figure 3).

 

Figure 3. Total alkaloid in the seed and stem of Telfairia occidentalis. Values with the

same letter superscript within the same constituent are not significantly (P < 0.05).

 

 

Antioxidant activity

The percentage antioxidant activity of the chloroform extract of both the seeds and stem extracts of T. occidentalis showed that the seeds had a higher antioxidant activity though not statistically significant. The antioxidant activity of the seed CHCl3 is mostly due to the presence of alkaloids present as shown in Figure 2. While that of the acetone might be due to the presence of cardiac glycoside, steroids and terpenoids as shown in Table 2.

 

The antioxidant activity of the acetone extract of the stem was greater than that of the seed though not statistically significant (Figure 4).  Antioxidant activity of plant extracts have been attributed to the flavonoids present in such plant extracts 27. Thus presence of flavonoid in the stem is responsible for its high antioxidant activity.

 

 

 

Figure 4. % DPPH scavenging activity at 0.5 mg/ml. Values with the same letter superscript within the same constituent are

 not significantly (P < 0.05).

 

 

 

 

CONCLUSION

The phytochemical analysis carried out on the seeds and stem extracts of Telfairia occidentalis showed the presence of phenolics, reducing sugars, cardiac glycosides, alkaloids, flavonoids, steroids and terpenoids.  The DPPH antioxidant assay showed a relatively good antioxidant activity with the acetone fraction of both the seed and stem extracts. These results confirm the folkloric claim of this plant.

 

ACKNOWLEDGEMENTS

The authors are grateful to the Technical staff of the department of Pharmaceutical and Medicinal Chemistry, University of Ilorin for their support.

 

REFERENCES

  1. Bodeker C, Bodeker G, Ong CK, Grundy CK, Burford G, Shein K (2005). WHO Global Atlas of Traditional, Complementary and Alternative Medicine. World Health Organization, Geneva.
  2. Mukherjee PW (2002). Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals. Business Horizons Publishers, New Delhi, India.
  3. WHO (2005). WHO Global Atlas of Traditional, Complementary and Alternative Medicine. World Health Organization, Geneva. 1and 2.
  4. Barnes J, Anderson LA, Phillipson JD (2007). Herbal medicine. 3rd Edition, Pharmaceutical Press, London. pp 1-23.
  5. Blumenthal M (2000) Ephedra update: industry coalition asks FDA to adopt national labeling guidelines on ephedra; offers co-operative research with NIH. Herbal Gram. 50: 64-65.
  6. Bisset NG (1994). Herbal Drugs and Phytopharmaceuticals. CRC Press, Boca Raton, FL.
  7. Bauer R (1998). Quality criteria and standardization of phytopharmaceuticals: Can acceptable drug standards be achieved? Drug Inform. Journal., 32: 101–110.
  8. Kayode AAA and Kayode OT (2011). Some Medicinal Values of Telfairia occidentalis: A Review. American Journal of Biochemistry and Molecular Biology. 1:30-38.
  9. Dina OA, Adedapo AA, Oyinloye OP and Saba AB (2006). Effect of Telfairia occidentalis extract on experimentally induced anaemia in domestic. African Journal of biomedical Research, 3: 181-183
  10. Oboh G, Nwanna EE and. Elusiyan CA (2006). Antioxidant and antimicrobial properties of Telfairia occidentalis (Fluted pumpkin) leaf extracts. Journal of Pharmacology and  Toxicology, 1: 167-175.
  11. Okokon JE, Ekpo AJ and Eseyin OA (2007). Antiplasmodial activity of ethanolic root extract of Telfairia occidentalis. Research Journal of Parasitology, 2: 94-98.
  12. Inuwa HM, Aimola IA, Muhammad A, Habila N, Okibe P, Latayo M, Ahmed Z (2012). Isolation and determination of omega-9 fatty acids from Telfairia occidentalis. International Journal of Food Nutrition and Safety,  1(1): 9-14.
  13. Eseyin OA, Ebong P, Ekpo A, Igboasoiyi A, Oforah E (2007). Hypoglycemic effect of the seed extract of Telfairia occidentalis in rat. Pakistan Journal of Biological Sciences, 10(3): 498- 501
  14. Danladi J, Abayomi KB, Dahiru AU (2012). Comparative Study of The Hepatoprotective Effect of Ethanolic Extract of a Telfairia occidentalis (Ugu) Leaves And Silymarin on Paracetamol Induced Liver Damage in Wistar Rats. International Journal of Animal and Veterinary Advances,  4(4): 235-239
  15. Cyril-Olutayo C, Agbedahunsi J, Elufioye O (2012). Antisickling properties of some indigenous and exotic plant species in Nigeria. Planta Medica, 78(11): I2- 17
  16. Sofowora A. (1996). Medicinal Plant and Traditional Medicine in Africa. 2nd Edn., Spectrum Books, Ibadan, Nigeria, pp: 112.
  17. Evans WC (1989). Trease and Evans’ Pharmacognosy. 13th edn, Bailliere Tindall, London. pp 15-57, 342-345, 480-517, 546 and 765-775
  18. Adedapo A, Jimoh F, Koduru S, Masika J and Afolayan A (2009). Assessment of the medicinal potentials of the methanol extracts of the leaves and stems of Buddleja salignaBMC Complementary and Alternative Medicine. 9:9-21
  19. Nabavi SM, Ebrahimzadeh M, Nabavi S, Hamidinia A and Bekhradnia AR (2008). Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica Mey. Pharmacol online. 2:560-567
  20. Harbone JB. And, Baxter H. Phytochemical Dictionary. A Handbook of Bioactive Compounds from Plants. London, Tytor and Francis Ltd. 1993; 70-127
  21. Deng Y, Yang G, Yue J, Qian B, Liu Z, Wang D, Zhong Y, Zhao Y (2014). Influences of ripening stages and extracting solvents on the polyphenolic compounds, antimicrobial and antioxidant activities of blueberry leaf extracts. Food Control. 38, 184–191.
  22. Eseyin OA, Sattar MA and Rathore HA (2014). A Review of the Pharmacological and Biological Activities of the Aerial Parts of Telfairia occidentalis Hook. f. (Cucurbitaceae). Tropical Journal of Pharmaceutical Research; 13:1761-1769
  23. Elemo BO, Elemo GN, Oladimeji OO and Komolafe YO (2002). Studies on the Composition of some Nutrients and Anti nutrients of Sheanut (Butyrospernum parkii). Nigerian Food Journal. 20: 69 – 73.
  24. Zamotaev IuN, Kremnev IuA and Podshibiakin SE (2005). Cardiac glycosides in complex treatment of patients with heart failure and supraventricular arrhythmias. Klinicheskaia Meditsina Journal. 83:59-63.
  25. Surovtseva YV, Jairam V, Salem AF, Sundaram RK, Bindra RS and Herzon SB (2016). Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay. Journal of the American Chemical Society. 138:3844-55
  26. Oyewole OA and Abalaka ME (2012). Antimicrobial Activities of Telfairia occidentalis (fluted pumpkins) Leaf Extract against Selected Intestinal Pathogens. Journal of Health Science.  2: 1-4
  27. Ndhlala AR, Finnie JF and Van Staden J (2010). In vitro antioxidant properties, HIV-1 reverse    transcriptase and acetylcholinesteras-e inhibitory effects of traditional herbal preparations sold in South Africa. Molecules. 15:6888-6904.