Faculty of Biotechnology, Ho Chi Minh City Open University,
Ho Chi Minh City, Vietnam

Corresponding author email: minh.np@ou.edu.vn

Article Publishing History

INTRODUCTION

Vernonia amygdalina commonly called bitter leaf is a perennial shrub belonging to the family Asteraceae (Ijeh and Ejike, 2011). It is a perennial shrub that is widely distributed in tropical region. It’s a great source of vitamins, minerals, polyphenols, alkaloids, saponins, flavonoids and steroids (Atangwho et al., 2009). This plant contains two sesquiterpene lactones: vernolide and vernodalol (Erasto et al., 2006). It is a versatile plant that possesses several prophylactic and therapeutic potential such as antidiabetic, anthelminthic, antiplasmodial, antimicrobial, antioxidant, antianaemic, immunomodulatory, cardiovascular properties (Atangwho et al., 2010; Saliu et al., 2012; Okolie et al., 2008; Akpaso et al., 2011; Ademola et al., 2011; Abay et al., 2015; Zofou et al., 2011; Omolola et al., 2013; Adesanoye et al., 2014; Oyeyemi et al., 2015; Momoh et al., 2012; Ezeonu et al., 2016; Abdulmalik et al., 2016). It is popularly consumed as a vegetable and is used  for medicinal purposes such as  to cure yellow fever, dysentry, constipation, malaria and stomach ache (Adegbite et al., 2009; Ebong et al., 2008, Suleiman et al., 2018).

In dehydration, the water content is decreased to a certain level where microbial proliferation will not happen while preserving the highest proximate composition. Medicinal and aromatic herbs are mostly maintained by air drying. Dehydrated products can be kept for a long storage and can be easily blended, powdered or packed for specific application or for deeper processing in the food or pharmaceutical purposes (Albert and Joachim, 2007). Infrared drying reveals several advantages compared to traditional drying. Infrared heating is faster than convective drying (Nowak & Lewicki, 2005). Infrared energy is transferred from the heating element to the sample, heating the material more rapidly and uniformly. Much more moisture emits from the irradiated surface and drying duration is shortened (Nowak & Lewicki, 2004). The leaf drying technique involves reducing moisture content of leaves to a point at which biochemical changes are limited while maintaining cell structure, pigment content and overall appearance (Singh and Dhaduk, 2005, Zaharaddeen and Samuel, 2019).

There are several notable studies mentioned to drying of  Vernonia amygdalina species. The drying behavior of Vernonia amygdalina leaves was investigated using open sun and shade drying. Open sun drying resulted in severe deformity of the leaf morphology which may lead to degradation of the phytochemicals (Oluwaseun et al., 2018). The effect of air, sun, oven and solar drying methods on the organic and dietary elemental composition of Vernonia amygdalina leaves was evaluated. Drying improved the concentration of both organic and dietary elemental components (Zaharaddeen and Samuel, 2019). Objective of this present study focused on the effectiveness of blanching, infrared drying power, temperature and velocity on total phenolic, flavonoid, DPPH, FRAP of dried herbal tea from Vernonia amygdalina leaves under the infrared irradiation.

MATERIAL AND METHODS

Vernonia amygdalina leaves were collected from Soc Trang province, Vietnam. After collecting, they must be kept in cool and dry cotton box, conveyed to laboratory for experiments. They were subjected to the blanching and infrared dehydration under different conditions. All standards and reagents such as Folin-Ciocalteu reagent, Na₂CO₃, gallic acid, Al(NO₃)₃, potassium acetate, DPPH, methanol, ethanol, acetate buffer, 2,4,6- tripyridyl-s-triazine, HCl , FeCl₃.6H₂O were analytical grade and purchased from Sigma-Aldrich. Lab utensils and equipments included weight balance, blender, infrared dryer, spectrophotometer.

Effect of blanching duration (s) to phytochemical and antioxidant properties of Vernonia amygdalina leaf: Raw Vernonia amygdalina leaves were blanched at 100^oC in different duration (15, 20, 25, 30, 35s). After being blanching, these blanched leaves would be would be dried by infrared dryer with drying power 40W at temperature 40^oC with air velocity 0.8 m/s. The dried samples were analyzed the total phenolic (mg GAE/100 g), flavonoid (mg QE/100 g), DPPH (%) and FRAP (mM TE/100g).

Effect of infrared drying power (W) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf: After finding the optimal blanching condition, these blanched leaves would be dried by infrared dryer under different powers (40, 80, 120, 160, 200 W) at temperature 40^oC with air velocity 0.8 m/s. After this experiment, the dried leaves would be analyzed the total phenolic (mg GAE/100 g), flavonoid (mg QE/100 g), DPPH (%) and FRAP (mM TE/100g).

Effect of infrared drying temperature (^oC) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf: By selecting the optimal steaming time and drying power, these blanched leaves would be dried by infrared dryer under power 120 W at different temperature (40, 45, 50, 55, 60^oC) with air velocity 0.8 m/s. After this experiment, the dried leaves would be analyzed the total phenolic (mg GAE/100 g), flavonoid (mg QE/100 g), DPPH (%) and FRAP (mM TE/100g)..

Effect of infrared drying air velocity (m/s) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf: By selecting the optimal steaming time, drying power and drying temperature, these blanched leaves would be dried by infrared dryer under power 120 W at temperature 50^oC with different air velocity values (0.8, 1.0, 1.2, 1.4, 1.6 m/s). After this experiment, the dried leaves would be analyzed the total phenolic (mg GAE/100 g), flavonoid (mg QE/100 g), DPPH (%) and FRAP (mM TE/100g).

Phytochemical and antioxidant estimation: Total phenolic content (mg GAE/100g) was evaluated using Folin–Ciocalteu assay (Nizar et al., 2014). Total flavonoid content (mg QE/100g) was evaluated by the aluminium calorimetric method (Formagio et al., 2015). The antioxidant activity was evaluated using DPPH (%) radical scavenging assay which was described by Huang et al. (2005). FRAP (mM TE/100g). FRAP (mM TE/100g) were performed according to Ivanov et al. (2014).

Statistical analysis: The experiments were run in triplicate with three different lots of samples. The data were presented as mean±standard deviation. Statistical analysis was performed by the Statgraphics Centurion version XVI.

RESULTS AND  DISCUSSION

Effect of blanching time (s) to the phytochemical and antioxidant properties of Vernonia amygdalina leaf: Blanching is an important step before drying of herbal materials to inactivate enzymes that cause browning and loss of bioactive constituents. Raw Vernonia amygdalina leaves were blanched by hot water at 100^oC in different durations (15, 20, 25, 30, 35s). After being blanching, these blanched leaves would be would be dried by infrared dryer with drying power 40W at temperature 40^oC in air velocity 0.8 m/s.  Results are presented in table 1.

It’s clearly noticed that 25s in blanching was adequate to maintain the highest amount of total phenolic, flavonoid, DPPH and FRAP. So this value was selected for further experiments. Omede et al. (2018) evaluated the antioxidant activity and cytotoxic properties of Vernonia amygdalina. They confirmed that the extracts possessed very low cytotoxicity (IC50 = 1.83 mg/ml), high total phenolic content (158.8 mg GAE/100g), total flavonoid (85.7 mg QE/100g).

Table 1. Effect of blanching time (s) to the phytochemical and antioxidant properties of Vernonia amygdalina leaf

Blanching duration (s)	15	20	25	30	35
Total phenolic (mg GAE/100g)	24.51±0.02b	26.30±0.01ab	29.47±0.02a	22.79±0.03bc	20.05±0.01c
Total flavonoid (mg QE/100g)	10.73±0.01b	12.64±0.00ab	14.55±0.03a	9.17±0.01bc	7.32±0.00c
DPPH (%)	31.50±0.00c	33.79±0.03b	36.04±0.01a	34.83±0.00ab	32.65±0.02bc
FRAP (mM TE/100g)	11.74±0.02c	13.01±0.01b	15.48±0.00a	14.84±0.02ab	12.36±0.03bc

^{Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%).}

Effect of infrared drying power (W) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf: Drying can lead to considerable loss of the available bioactive components due to thermal degradation depending on the drying method and temperature conditions (Naomi et al., 2018).

They proved that total phenolic content, antioxidant capacity were significantly affected by drying method and drying temperature. By finding the optimal blanching condition, these blanched leaves would be dried by infrared dryer under different powers (40, 80, 120, 160, 200 W) at temperature 40^oC with air velocity 0.8 m/s. Our results were presented in table 2. It’s clearly noted that the optimal infrared drying power should be 160 W to preserve the best phytochemical and antioxidant attributes of dried Vernonia amygdalina leaf. So this value was selected for further experiments.

Table 2. Effect of infrared drying power (W) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf

Drying power (W)	40	80	120	160	200
Total phenolic (mg GAE/100g)	29.47±0.02c	33.49±0.03bc	38.64±0.01a	36.12±0.00ab	34.41±0.03b
Total flavonoid (mg QE/100g)	14.55±0.03c	15.74±0.01bc	17.63±0.03a	17.05±0.02ab	16.54±0.00b
DPPH (%)	36.04±0.01bc	38.61±0.00ab	40.82±0.02a	37.53±0.01b	33.71±0.02c
FRAP (mM TE/100g)	15.48±0.00c	17.01±0.02b	19.26±0.00a	18.95±0.03ab	16.58±0.01bc

^{Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%).}

Effect of infrared drying temperature (^oC) the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf: Infrared irradiation has potential for drying herbs because it is gentle and shortens the processing duration. The leaf drying is an important post-harvest strategy for improving product quality as well as providing added value. By selecting the optimal steaming time and drying power, these blanched leaves would be dried by infrared dryer under power  120W at different temperature (40, 45, 50, 55, 60^oC) with air velocity 0.8 m/s.

Our results are shown  in table 3. The optimal drying temperature was recorded at 50^oC to preserve the best phytochemical and antioxidant attributes of dried Vernonia amygdalina leaf  so we choose this value for further experiments. In one report, Oluwaseun et al. (2018) proved that shade drying was the better way of drying V. amygdalina leaves to preserve the nutrients compared to open sun drying.

Table 3. Effect of infrared drying temperature (^oC) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf

Drying temperature (oC)	40	45	50	55	60
Total phenolic (mg GAE/100g)	38.64±0.01c	39.14±0.03bc	40.53±0.02a	40.09±0.03ab	39.64±0.00b
Total flavonoid (mg QE/100g)	17.63±0.03c	18.25±0.01bc	19.78±0.00a	19.33±0.01ab	18.96±0.03b
DPPH (%)	40.82±0.02c	41.98±0.00ab	42.56±0.01a	41.32±0.02b	41.07±0.00bc
FRAP (mM TE/100g)	19.26±0.00c	20.76±0.02bc	21.83±0.03a	21.35±0.00ab	21.01±0.01b

^{Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%).}

Effect of infrared drying air velocity (m/s) the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf: By selecting the optimal steaming time, drying power and drying temperature, these blanched leaves would be dried by infrared dryer under power 12 W at temperature 50^oC with different air velocity values (0.8, 1.0, 1.2, 1.4, 1.6 m/s). Our results are shown in table 4.

The optimal air velocity was recorded at 1.4 m/s to preserve the best phytochemical and antioxidant attributes of dried Vernonia amygdalina leaf so we choose this value for application. In one study, Zaharaddeen and Samuel (2019) proved that oven–solar drying method could be useful in preserving Vernonia amygdalina leaves in a more hygienic way and ensure its all-the-year round availability and possibly elimination of most nutrient deficiencies.

Table 4. Effect of air drying velocity (m/s) to the phytochemical and antioxidant properties of dried Vernonia amygdalina leaf

Air drying velocity (m/s)	0.8	1.0	1.2	1.4	1.6
Total phenolic (mg GAE/100g)	40.53±0.02b	40.98±0.01ab	41.25±0.03ab	41.39±0.00a	41.43±0.01a
Total flavonoid (mg QE/100g)	19.78±0.00b	20.03±0.03ab	20.27±0.01ab	20.88±0.02a	20.91±0.03a
DPPH (%)	42.56±0.01b	42.88±0.02ab	42.97±0.00ab	43.31±0.03a	43.36±0.02a
FRAP (mM TE/100g)	21.83±0.03b	22.01±0.00ab	22.34±0.02ab	22.65±0.00a	22.69±0.01a

^{Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%)}

CONCLUSION

V. amygdalina contains numerous phytochemical constituents associating with its potent pharmacological properties to treat various ailments. We have successfully examined different technical variables influencing to the blanching and drying process of Vernonia amygdalina leaf. Due to low moisture content, these dried leaves can be kept in ambient environment for longer periods without losing their appearance and pharmaceutical value. V. amygdalina would be a safe potential source of natural antioxidant agent as a functional food.

REFERENCES

Abay SM, Lucantoni L, Dahiya N, Dori G, Dembo EG, Esposito F, Lupidi G, Ogboi S, Ouédraogo RK, Sinisi A, Taglialatela Scafati O(2015). Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds. Malaria Journal 14: 288.

Abdulmalik O, Oladapo OO, Bolaji MO (2016) Effect of aqueous extract of Vernonia amygdalina on atherosclerosis in rabbits. ARYA Atherosclerosis 12: 35.

Adegbite AE, Sanyaolu EB. (2009) Cytotoxicity testing of aqueous extract of bitter leaf (Vernonia amygdalina Del.) using the Allium cepa chromosome aberration assay. Scientific Research and Essays  4: 1311-1314.

Ademola IO, Eloff JN. (2011) Anthelminthic activity of acetone extract and fractions of Vernonia amygdalina against Haemonchus contortus eggs and larvae. Tropical Animal Health and Production 43: 521-527.

Adesanoye OA, Farombi EO.(2014) In vitro Antioxidant Properties of methanolic leaf extract of Vernonia amygdalina Del. Nigerian Journal of Physiological Sciences 2014; 29: 93-101.

Akpaso MI, Atangwho IJ, Akpantah A, Fischer VA, Igiri AO, Ebong PE. Effect of combined leaf extracts of Vernonia amygdalina (Bitter Leaf) and Gongronema latifolium (Utazi) on the pancreatic [beta]-cells of Streptozotocin-induced diabetic rats. British Journal of Medicine and Medical Research 2011; 1: 24.

Albert GWH and Joachim M.(2007) Microwave drying of medicinal and aromatic plants. Stewart Postharvest Review 4: 1-6.

Atangwho IJ, Ebong PE, Eyong EU, Williams IO, Eten MU, Egbung GE (2009). Comparative chemical composition of leaves of some antidiabetic medicinal plants: Azadirachta indica, Vernonia amygdalina and Gongronema latifolium. African Journal of Biotechnology 8: 18.

Atangwho IJ, Ebong PE, Egbung GE, Obi AU.(2010) Extract of Vernonia amygdalina Del. (African bitter leaf) can reverse pancreatic cellular lesion after alloxan damage in the rat. Australian Journal of Basic and Applied Sciences  4: 711-716.

Ebong PE, Atangwho IJ, Eyong EU, Egbung GE. (2008) The antidiabetic efficacy of combined extracts from two continental plants: Azadirachta indica (A. Juss) (Neem) and Vernonia amygdalina (Del.) (African bitter leaf). American Journal of Biochemistry and Biotechnology  4: 239-244

Erasto P, Grierson DS, Afolayan AJ.(2006) Bioactive sesquiterpene lactones from the leaves of Vernonia amygdalina. Journal of Ethnopharmacology 2006; 106: 117-20.

Ezeonu IM, Asuquo AE, Ukwah BN, Ukoha PO.(2016) Immunomodulatory properties of prebiotics extracted from Vernonia amygdalina. African Journal of Traditional, Complementary and Alternative Medicines 2016; 13: 11-7.

Formagio ASN, Ramos DD, Vieira MC, Ramalho SR, Silva MM, Zárate NAH, Foglio MA, Carvalho JE.(2015) Phenolic compounds of Hibiscus sabdariffa and influence of organic residues on its antioxidant and antitumoral properties. Braz. J. Biol. 2015; 75: 69-76.

Huang D, Ou B, Propr RL. (2005) The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry 2005; 53: 1841-1856.

Ivanov IG, Vrancheva RZ, Marchev AS, Petkova NT, Aneva IY, Denev PP, and Pavlov AI. (2014) Antioxidant activities and phenolic compounds in Bulgarian Fumaria species. International Journal of Current Microbiology and Applied Sciences  3: 296-306.

Ijeh II, Ejike CE.(2011)  Current perspectives on the medicinal potentials of Vernonia amygdalina Del. Journal of Medicinal Plants Research 2011; 5: 1051-1061.

Momoh MA, Muhamed U, Agboke AA, Akpabio EI, Osonwa UE.(2012) Immunological effect of aqueous extract of Vernonia amygdalina and a known immune booster called immunace® and their admixtures on HIV/AIDS clients: a comparative study. Asian Pacific Journal of Tropical Biomedicine  2: 181-184.

Naomi MN, Willis OO, Jane A, Daniel SN.(2018)  Effect of drying methods on the retention of bioactive compounds in African eggplant. Food Sci. Nutr.  6: 814-823.

Nizar S, Elhadi MM, Algaili MA, Hozeifa MH, Mohamed O.(2014)  Determination of total phenolic content and antioxidant activity of roselle (Hibiscus sabdariffa L.) calyx ethanolic extract. Standard Research Journal of Pharmacy and Pharmacology 2014; 1: 034-039.

Nowak D, Lewicki PP.(2004) Infrared drying of apple slices. Innovative Food Science & Emerging Technologies 2004; 5: 353–360.

Okolie UV, Okeke CE, Oli JM, Ehiemere IO. (2008)Hypoglycemic indices of Vernonia amygdalina on postprandial blood glucose concentration of healthy humans. African Journal of Biotechnology  7: 24.

Oluwaseun R, Nour HA, Siti KAM, Olusegun AO. (2018) Mathematical modeling of thin layer drying using open sun and shade of Vernonia amygdalina leaves. Agriculture and Natural Resources  52: 53-58.

Omede A, Suleiman MS, Atanu FO, Sheneni VD, Jegede ER.(2018) Evaluation of antioxidant and cytotoxic properties of Vernonia amygdalina. International Journal of Plant studies  1: 1-6.

Omolola AA, Olorunfemi RM, Adetutu AD, Adeniyi SA, Ebenezer OF,(2013) Modulatory effect of methanolic extract of Vernonia amygdalina (MEVA) on tert-butyl hydroperoxide– induced erythrocyte haemolysis. Cell Biochemistry and Function  31: 545–550.

Oyeyemi MO, Soetan KO, Akinpelu OB.(2015) Sperm characteristics and haemogram of male albino rats (wistar strain) treated with saponin extract from Vernonia amygdalina Del. asteraeceae 110192. Journal of Cell and Animal Biology  9: 26-30.

Saliu JA, Ademiluyi AO, Akinyemi AJ, Oboh G.(2012) In Vitro antidiabetes and antihypertension properties of phenolic extracts from bitter leaf (Vernonia amygdalina DEL.) Journal of Food Biochemistry   36: 569–576.

Singh A and Dhaduk BK.(2005) Effect of dehydration techniques in some selected leaves. J. Orna. Hort 8: 155-156.

Suleiman D, Mohammed AH, Idris AM, Umar II.(2018) Vernonia amygdalina Del: A mini review. Research J. Pharm. and Tech. 11: 4187-4190.

Zaharaddeen NG and Samuel O (2019). The effect of different drying methods on the elemental and nutritional composition of Vernonia amygdalina (bitter leaf). Journal of Taibah University for Science 2019: 13: 396–401.

Zofou D, Tene M, Ngemenya MN, Tane P, Titanji VP.(2011) In vitro antiplasmodial activity and cytotoxicity of extracts of selected medicinal plants used by traditional healers of Western Cameroon. Malaria Research and Treatment  12: 2011.