vernonia amygdalina leaf: unveiling its antacid and...

ISSN: 2410-8790 Mbatchou et al / Current Science Perspectives 3(3) (2017) 148-155 iscientic.org.

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Article type: Research article Article history: Received March 2017 Accepted July July 2017 Issue Keywords: Vernonia amygdalina leaf Gastrone® In vitro Antacid Carminative properties.

Leaves of Vernonia amygdalina commonly known as bitter leaf is widely consumed in the sub-Saharan regions of Africa. This study therefore considers the properties of leaves of the plant as alternative sources of an antacid and carminative drug. Bioactive compounds were extracted separately with methanol and distilled water. The neutralizing capacity, duration of neutralizing effects and amounts of carbon dioxide evolved in the antacid and carminative studies respectively were determined for both aqueous and methanol leaf extracts. Aqueous leaf extract showed a significant antacid and carminative potential (P < 0.05) when compared with the methanolic leaf extract at all the tested concentrations. The highest neutralizing effect and duration of neutralizing effect obtained for the aqueous leaf extract were 2.24 ± 0.000 and 162.56 ± 0.087 minutes respectively as against 1.97 ± 0.007and 95.55 ± 0.083 minutes of the methanolic leaf extract. Further evidence is provided in the high neutralizing capacity of the aqueous leaf extract (34.93 ± 0.088mL) compared to that of the methanolic extract (7.80 ± 0.115mL). Higher doses of the aqueous leaf extract were however needed for comparable results with the control Gastrone® (13.5252g of aqueous extract ≡ ~3.1582g of Gastrone® (antacid); 5.3797g of aqueous extract ≡ ~3.1582g of Gastrone® (carminative). Scientific data in this research work therefore supports the local use of aqueous leaf extract of Vernonia amygdalina as an antacid and carminative agent by the people of Tamale, Northern region, Ghana.

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Capsule Summary: Aqueous and methanol extracts of the secondary metabolites of Vernonia amygdalina leaf exhibited variable antacid and carminative activities

Cite This Article As: Valentine Chi Mbatchou, Kanwugu Osman Nabayire and Yesman Akuoko. Vernonia amygdalina Leaf: Unveiling its antacid and carminative properties In Vitro. Current Science Perspectives 3(3) (2017) 148-155

INTRODUCTION

Hyperacidity also known as acid dyspepsia is the commonest gastrointestinal malady caused by excessive amounts of hydrochloric acid in the stomach secreted by the

parietal cells. It is caused by various factors ranging from a bacterial infection (Helicobacter pylori) to eating habit and lifestyle (Nanjaiah et al., 2013). Hyperacidity is associated with a number of discomforting conditions including heartburns, formation of gas in stomach, epigastric pain, postprandial fullness, anorexia, inflammation of the stomach

Current Science Perspectives 3(3) (2017) 148-155

Vernonia amygdalina Leaf: Unveiling its antacid and carminative properties In Vitro

Valentine Chi Mbatchou1*, Kanwugu Osman Nabayire1 and Yesman Akuoko2

1Department of Applied Chemistry and Biochemistry, University for Development Studies (UDS), Navrongo, Ghana 2Department of Chemistry, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi. Ghana

*Corresponding author’s E-mail: [email protected]

A R T I C L E I N F O A B S T R A C T

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lining (Ingale et al., 2014; Ankit et al., 2014) and major clinical conditions such as ulceration (gastric and duodenal) and gastro-esophageal reflux disease (GERD) (Linares et al., 2014; Sandhya et al., 2012). GERD affects millions of people worldwide and has emerged as an important and common acid related disorder, affecting approximately 35-40% of adults in the western world with 36% of them having symptoms at least once a month, and millions of people worldwide (Robinson et al., 2002; Tytgat and Simoneau, 2006; Klochan and Vaezi, 2010). Peptic ulcer (PU) or peptic ulcer disease (PUD), defined as a break in the mucosal lining of the gastrointestinal tract (GIT), remains a relatively common gastrointestinal disorder and is induced by factors such as stress, smoking, nutritional deficiencies, H. pylori infection and ingestion of non-steroidal anti-inflammatory drugs (NAIDs) (Fallone and Mayrand, 2001; Mukherjee et al., 2010). PU/PUD is a common cause of acute upper gastrointestinal bleeding whose incidence ranges from 36 to 172 per 100000 populations (Wong and Chan, 2010).

Acid suppression continues to be the main medical strategy to treat acid related diseases and for decades, millions of patients have had their gastric neutralized (with antacids) or effectively inhibited with histamine-2 receptor antagonists (H2RAs) and proton pump inhibitors (PPIs). Antacids work almost instantaneously and are therefore used for rapid and effective relief of mild or intermittent symptoms. The common active ingredients in the currently available antacids consist of either one or a combination of the following: magnesium hydroxide, aluminum hydroxide, calcium carbonate, sodium bicarbonate and magnesium carbonate (Orwa et al., 2012; Meija and Kraft, 2009). In recent times, intensive antacid therapy is often not acceptable. This is because the duration of neutralization effect exhibited by antacids is short and also due to the side effects (such as diarrhoea, constipation, interference with drug absorption and rarely, renal, metabolic and acid-base disturbance) exhibited by these synthetic antacids (Schubert and Peura, 2008; Aihara et al, 2003). Formation of gases in

Table 1: Organoleptic properties of Vernonia amygdalina leaf extracts.

Extracts Smell Colour Texture Percentage yield (%w/w)

Methanol extract Pungent Green Crystalline 5.32 Aqueous extract Pungent Brown Sticky 9.83

Table 2: Phytochemicals of Vernonia amygdalina leaf extracts.

Phytochemicals Methanol extract Aqueous extract

Saponins + +

Tannins + +

Flavonoids + -

Steroids + -

Terpenoids + +

Alkaloids + +

Cardiac glycosides - + - ≡ Absent, + ≡ Present

Table 3: Antacid activity of Vernonia amygdalina leaf extracts.

Extracts/ Controls

Conc. (mg/90mL)

Temperature Effect on pH (25

oC -37

oC)

Neutralizing Effect

Duration of Neutralizing Effect (min)

Amount of Artificial Gastric acid Consumed (mL)

No of H+ ions

consumed (mmol)

Methanol 250 6.81 – 6.89 1.85 ± 0.007٭0.009 ±0.32 ٭0.115 ± 5.00 ٭0.090 ± 79.63 ٭

٭0.003 ± 0.40 ٭0.067 ± 6.27 ٭0.035 ± 87.57 ٭0.007 ± 1.91 4.82 – 4.72 500

٭0.006 ± 0.49 ٭0.115 ± 7.80 ٭0.083 ± 95.55 0.007 ± 1.97 3.96 – 3.92 750Aqueous 250 6.64 – 6.71 2.09 ± 0.007٭0.006 ± 0.86 ٭0.088 ± 13.63 ٭0.133 ± 115.53 ٭

٭0.003 ± 1.59 ٭0.067 ± 25.27 ٭0.088 ± 144.53 ٭0.000 ± 2.22 6.42 – 6.38 500 ٭0.003 ± 2.20 ٭0.088 ± 34.93 ٭0.087 ± 162.56 ٭0.000 ± 2.34 6.30 – 6.26 750

NaHCO3 (Control 1)

٭0.003 ± 2.80 ٭0.058 ± 44.40 ٭0.091 ± 203.87 ٭0.003 ± 1.94 8.36 – 8.32 250 ٭0.006 ± 5.86 ٭0.088 ± 92.87 ٭0.180 ± 330.45 ٭0.007 ± 2.31 8.51 – 8.39 500 ٭0.028 ± 8.88 ٭0.437 ± 140.67 ٭0.188 ± 492.33 ٭0.007 ± 2.73 8.65 – 8.54 750

Gastrone® (Control 2)

٭0.000 ± 2.71 ٭0.033 ± 42.93 ٭0.219 ± 173.52 ٭0.012 ± 1.82 8.15 – 8.04 250 ٭0.003 ± 5.63 ٭0.058 ± 89.32 ٭0.038 ± 300.65 ٭0.007 ± 1.93 8.31 – 8.25 500 ٭0.000 ± 8.57 ٭0.033 ± 135.83 ٭0.064 ± 460.97 ٭0.007 ± 2.15 8.57 – 8.46 750

Water (Control 3)

90 5.28 – 6.11 1.98 ± 0.000 76.08 ± 0.020 1.43 ± 0.033 0.09 ± 0.000

Results are expressed as mean ± SEM

P > 0.05 when compared with distilled water using the Dunnett Multiple Comparison Test.





the stomach is usually due to incomplete digestion of carbohydrates but however occurs during hyperacidity. It also occurs as a metabolic by-product of some foods. Carminatives are the agents which induce the expulsion of gas from the stomach or intestines (Wu and Chen, 2010).

Plant species over the years have served as attractive sources of new drugs. Vernonia amygdalina, commonly known as bitter leaf plant is a rapid regenerating soft wooded shrub known to be indigenous to a variety of ecological zones in Africa especially sub-Sahara regions. Various parts of the plant have been used as therapeutic agents in disease conditions including gastrointestinal problems, microbial and parasitic infections, hepatoxities, diabetes and cancer (Agbogidi and Akpomorine, 2013; Adediran et al., 2014). Its leaf is a soup vegetable which is being used in Nigeria and also the northern part of Ghana for centuries (Utoh-Nedosa, 2010). The potential of Vernonia amygdalina as an antacid and carminative has however not been exploited scientifically hence this research seeks to explore the antacid and carminative properties of the leaves of Vernonia amygdalina.

MATERIAL AND METHODS

Chemicals

NaHCO3, Gastrone® and gastric acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Methanol were obtained from Merck Chemical Supplies (Damstadt, Germany). All reagents were of analytical grade.

Sample collection and preparation

Leaves of Vernonia amygdalina were collected within the environs of Navrongo in the Upper East region of Ghana and authenticated at the Department of Applied Biology, University for Development Studies (UDS). A Voucher specimen was deposited at the herbarium of the Department of Applied Biology (UDS). The fresh leaves were rinsed with distilled water, de-stemmed then chopped into smaller dimensions and air dried for four days at room temperature

(25 °C). The dried leaves were ground into fine powder using an electric blender and stored in an air-tight container.

Extraction of phytochemicals

Extraction was carried out by employing the method outlined by Ajetunmobi and Towolawi (2014) with some slight modifications. Extraction time for extracts was extended from 48 to 72 hours. Two hundred grams (200g) of the dried leaf powder were separately soaked in 1000mL of methanol and distilled water in 2.5L amber colored bottles for 72hrs. The percolates were then collected and concentrated at room temperature to obtain dried extracts and the percentage yields (% w/w) calculated.

Qualitative phytochemical screening

Saponins and cardiac glycosides were evaluated using protocols described by Mbatchou et al. (2011), Ajetunmobi and Towolawi (2014) respectively while tannins, flavonoids, alkaloids, steroids and terpenoids were evaluated using protocols by Ankita et al. (2012).

Preparation of extract concentrations

Three concentrations (250mg/90mL, 500mg/90mL and 750mg/90mL) of each extract (methanol and aqueous extracts) were prepared and used for the various tests. Concentrations were always prepared fresh whenever needed. Similar concentrations of active sodium bicarbonate and Gastrone® were prepared to serve as positive controls.

Preparation of artificial gastric acid

Artificial gastric acid was prepared using protocols described by Vandana and Prashant (2013).

Determination of effect of temperature on pH of Extract Concentrations

pH of various concentrations of the leaf extracts were determined at temperatures ranging from 25 – 37oC. pH

Table 4: Amounts of CO2 expelled by Vernonia amygdalina leaf extracts.

Extract/Controls Concentration (mg/90mL) Amount of CO2 (mmol)

Methanol extract 250 0.40 ± 0.000 ٭0.007 ± 0.83 500 ٭0.000 ± 1.25 750

Aqueous extract 250 0.48 ± 0.017٭ ٭0.010 ± 1.02 500 ٭0.017 ± 1.51 750

NaHCO3 (Control 1) 250 0.65 ± 0.029٭ ٭0.015 ± 1.38 500 ٭0.083 ± 1.78 750

Gastrone® (Control 2) 250 0.87 ± 0.033٭ ٭0.007 ± 1.73 500 ٭0.015 ± 2.68 750

Water (control 3) 90 0.31 ± 0.010 Results are expressed as mean ± SEM

.p > 0.05 when compared with distilled water using the Dunnett Multiple Comparison Test ٭





values of NaHCO3, Gastrone® and water also determined for comparison (Each procedure was done in triplicates for each test solution).

Determination of Neutralizing Effect on Artificial Gastric Acid

The neutralizing effect of the different concentrations were determined according to protocols described by Vandana and

Fig. 1: Effect of temperature on the pH of leaf extracts.

Fig. 2: Effect of temperature on the pH of controls.





Prashant (2013) with NaHCO3, Gastrone® and water as controls. (Each procedure was conducted in triplicates for each test solution).

Determination of the Duration of Neutralizing Effect on Artificial Gastric Acid

A 50mL burette was mounted on a retort stand and filled with artificial gastric acid then set at a flow rate of 3mL/min. 90mL of freshly prepared extract concentration was introduced in a 250mL beaker already containing 100mL of artificial gastric acid at a temperature of 37oC and pH equals 1.2. The resulting solution was swirled continuously as the artificial gastric acid was added from the burette maintaining the flow rate at 3mL/min. A pH meter was attached to the beaker to continuously monitor the changes in pH and the time taken to return to pH of 1.2 was noted. The temperature

of the resulting solution in the beaker was maintained at 37oC throughout the experiment. Each experiment were carried out in triplicates for the different extract concentrations, water, sodium bicarbonate and Gastrone®.

Determination of the Neutralization Capacity

The neutralizing capacity was determined according to the method described by Vandana and Prashant (2013) with NaHCO3, Gastrone® and water as controls. (Each procedure was done in triplicates for each test solution). The total H+ consumed was calculated as:

(H+) (mmol) = 0.063096 (mmol/mL) × V (mL).

Carminative Activity Studies

The carminative potential of the leaf extract was determined for 250mg, 500mg and 750mg of the leaf extract according to the protocol outlined by Swapnil et al. (2012) with a slight modification. The expelled carbon dioxide was trapped in a different Erlenmeyer flask connected to the flask containing the extract via a tube. The number of moles of carbon dioxide expelled was calculated and tabulated.

RESULTS AND DISCUSSION

Phytochemical screening of the leaf extracts revealed positive results for all tested phytochemicals with the exception of cardiac glycoside in the case of the methanolic extract and flavonoids and steroids in the case of the aqueous extract. The effect of temperature on the pH of leaf extracts showed the pH of the extracts as well as that of NaHCO3 and Gastrone® varying slightly within the tested temperature range (25°C to 37°C), wide variations were however observed for distilled water. The leaf extracts and the NaHCO3 and Gastrone® exhibited impressive thermo-stability from room temperature to body

temperature. Results indicate that with the exception of the methanolic leaf extract, all treatments possessed significant (P < 0.05) gastric neutralizing effect in comparison with distilled water (1.98 ± 0.000). Comparing leaf extracts at the highest tested concentration with NaHCO3 (2.73 ± 0.007) and Gastrone® (2.15 ± 0.007), as it can be seen from Fig. 3, the aqueous extract exhibited a promising neutralizing effect by increasing the pH of the artificial gastric acid from 1.2 to 2.24 ± 0.000 as compared to 1.97 ± 0.007 produced by the methanolic leaf extract. With regards to the duration of neutralizing effect, all treatments including methanolic leaf extract, aqueous leaf extract, NaHCO3 and Gastrone® showed significantly longer duration when compared with water (76.08 ± 0.020 minutes). NaHCO3 gave the longest duration (203.87 ± 0.091, 330.45 ± 0.180 and 492.33 ± 0.188 minutes) at all tested concentrations followed by Gastrone® then the aqueous leaf extract with the methanolic leaf extract giving

Fig. 3: A bar chart representing the neutralizing capacity of leaf extracts and control.

Fig. 4: A linear plot of the neutralizing capacity of aqueous leaf extract and control.





the least (79.63 ± 0.090, 87.57 ± 0.035, 95.55 ± 0.083minutes) at the same concentrations. The neutralizing effect and duration were thus dose dependent. Also, the leaf extracts, NaHCO3 and Gastrone® exhibited significant acid neutralizing capacity when compared with water (P < 0.05). The neutralizing capacity of the leaf extracts, NaHCO3 and Gastrone® were therefore dose dependent. The antacid ability of the aqueous leaf extract were significantly higher

relative to the methanolic leaf extract (P < 0.05). The highest neutralizing effect and duration of neutralizing effect obtained for the aqueous leaf extract were 2.24 ± 0.000 and 162.56 ± 0.087minutes respectively. These values were greater than those obtained for the methanolic leaf extract (1.97 ± 0.007and 95.55 ± 0.083minutes). Thus, the aqueous extract presented a better potential as an antacid than the methanolic extract. Further evidence is provided in the high

Fig. 5: A bar chart representing amounts of CO2 expelled by leaf extracts and control.

Fig. 6: A line graph of the carminative activity of aqueous leaf extract and Gastrone®.





neutralizing capacity of the aqueous leaf extract (34.93 ± 0.088mL) compared to that of the methanolic extract (7.80 ± 0.115mL). However, when compared with the controls NaHCO3 and Gastrone® at similar concentrations, the leaf extracts exhibited minimal antacid activity owing to the crude nature of the leaf extracts. The minimum recommended dose of Gastrone® per the drug leaflet is two (2) tablets (~3.1582g) or 10mL of the suspension and consumes a total of 36.72734mmoles of H+ ions. To produce similar effects, the amount of aqueous crude leaf extract needed was determined to be 13.5252g an indication of higher amounts of the crude extract being needed to produce matching effect as Gastrone. All treatments possessed significant carminative potential when compared with water (P > 0.05). At all the amounts tested (250, 500 and 750mg), Gastrone® demonstrated the highest carminative potential followed by NaHCO3. Both methanolic and aqueous leaf extracts exhibited comparable carminative potentials with the aqueous leaf extract producing a better result. The recommended minimum dose of 5.3797g of the aqueous extract is equivalent to that of Gastrone® (2 tablets, ~3.1582g) which expels 11.31952mmol of carbon-dioxide and a positive indication of the aqueous leaf extract possessing very promising carminative ability. CONCLUSIONS Vernonia amygdalina leaf extracts displayed diverse phytochemicals accounting for its observed antacid and carminative properties. In all accounts, the aqueous extract exhibited greater neutralizing effect and duration than the methanolic extract. Though both extracts showed promising antacid and carminative properties, that of the aqueous extract at all concentrations were more prominent. The leaves of Vernonia amygdalina therefore provide an alternative means to the treatment of hyperacidity and management of PU/PUD and GERD. Furthermore, the potential of this plant species as an antacid and a carminative agent is of great interest and warrants further studies. ACKNOWLEDGEMENTS The authors of this work are grateful to Dr. Isaac Sackey of the Department of Applied Biology, University for Development Studies for authentication of the plant investigated and also to the entire staff of the Department of Applied Chemistry and Biochemistry of University for Development Studies for their support, most especially Mr. Daniel Oduro Mensah for providing the enzyme pepsin which was used to prepare artificial gastric acid used in this study. CONFLICT OF INTEREST The authors declare that there are no conflict of interest with other people or organizations. REFERENCES

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vernonia amygdalina leaf: unveiling its antacid and...

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