Screening of Phytochemicals and Comparative Antioxidant activity of Leaf and Fruit of MalaysianMengkudu Using Aqueous and Organic Solvent Extracts.

 

U.S MahadevaRao¹*, Khamsah Suryati Mohd², Siti Zulaikha Bt Abd Halim², Masitah Bt Khamis²

¹Faculty of Medicine and Health Science, Universiti Sultan ZainalAbidin, Malaysia.

²Faculty of Agriculture, Biotechnology and Food Science, Universiti Sultan Zainal Abidin , Malaysia.

*Corresponding Author E-mail: raousm@gmail.com; raousm@unisza.edu.my

 

Background: Ayurveda deals with the propensity of the herb to bestow upon the body enhanced capacity of warding off infections and delaying the ageing progression. Ethnic natural products have become the substitute way to switch the synthetic medicine. Aim: An endeavor has been made to evaluate the phytochemicals and free radical scavenging efficacy of different parts of Malaysian Mengkudu using different solvent extracts. Methods: The herbal powder obtained from various parts of Mengkuduplant i.e. dry leaves, dry fruits and fresh fruits were extracted with various solvents. The extracts were analyzed for phytochemicals and antioxidants, carotenoids, ascorbic acid, tocopherol, total phenol, proteins, reducing sugars and sterols. Antioxidant activity was reconnoiteredin terms of superoxideradical scavenging assay and reducing power assay. Result: Phytochemical characterization of thedifferent extracts revealed the presence of the phytochemicals-alkaloids, coumarin, phenols, flavonoids, sterol, saponin glycosides, reducing sugars, proteins, cardio active aglycones and cardinolides. Discussion: Excellent superoxide radical scavenging ability found in almost all extracts of Malaysian Mengkudu. In the present study superoxide radical reduces nitroblue tetrazolium(NBT) to a blue colored formazan that is measured at 560 nm. Antioxidant activity has been reported to be concomitant with development of reducing power. This shows that extracts might comprise reductones like ascorbic acid, reducing sugar, thiol group encompassing protein which could react with free radicals to alleviate and sack the radical chain reaction. Conclusion: These outcomes propose that the guaranteeing phytonutrients of the plant could be subjugated against oxidative stress, cancer, ageing, ischemic heart disease in dissolving thrombus, microbial infections and hormone replacementtherapy (HRT) justifying their use in conventional medicine as nutraceuticals.

 

 

 

INTRODUCTION:

The use of medicinal plants as source of the therapies for the treatment of many infirmitiesepochs back to prehistory and people of all continents have this old tradition^[1]. In developing countries where medicines are rather expensive, it is profound that these medicinal plants will find their way in the arsenal of antimicrobial drugs ^[2]. Phytochemical evaluation plays an important role in the standardization of crude herbal drugs ^[3]. Limited data from animal studies submits that very high intake of phytosterols, particularly sitosterol, may inhibit the growth of breast and prostate cancer ^[4-6]. Indole alkaloids exhibit numerous biological activities such as anti-tumor, anti-microbial, anti-hypertensive and central nervous system stimulant ^[7].

 

A large body of scientific evidence associating dietary phytochemicals with health and well-being of population has stimulated fabulous accomplishments to develop and commercialize products variously recognized as nutraceuticals, phytoceuticals, dietary supplements, functional foods, etc.^[8]. During 1980s and 1990s, numerous laboratories began studying phytochemicals to “mine” plants for bioactive substances that might be used as drugs (nutraceuticals) or for other chemical applications. Many compounds are showing prodigious promise as disease fighters in the body, furthering production or activities of enzymes, which then act by stalling carcinogens, subduing malignant cells, or snooping with the processes that can cause heart disease and stroke ^[9].

 

In the last few decades, several epidemiological studies have shown that a dietary intake of foods rich in natural antioxidants correlates with reduced risk of coronary heart disease particularly; a negative association between consumption of polyphenol-rich foods and cardiovascular diseases has been demonstrated. This association has been moderately explicated on the basis of the fact that that polyphenols interject lipid peroxidation induced by reactive oxygen species (ROS) ^{[10, 11]}. Different incitements, leading to an increase of ROS generation inside the cell, galvanizethe phosphorylation of IkB inhibitory protein and the subsequent proteolysis. Thioredoxin may reduce activated NF-kB proteins facilitating nuclear translocation. Once released from IkB, the NF-kB complex translocates into the nucleus and the binding to DNA domain in the promoters and enhancers of genes such as TNF-a, IL-1, proliferation and chemotactic factors, adhesion molecule. Some of these genes, in turn, may further induce NF-kB activation, leading to a vicious circle if the regulatory cellular system escapes from control. Polyphenolic compounds as natural phytochemical antioxidants that possess anti-inflammatory properties by down regulating NF-kB^[12-14].

 

A dramatic shift has taken place in consumer attitudes toward food. An increasingly active population, the pursuit of healthier lifestyles and the desire to live longer has given rise to a new category of food products referred to as “nutraceuticals.” Incorporated in the nutraceuticals category are functional foods. Functional foods can be described as conventional foods containing ingredients that provide additional health or nutritional benefits beyond basic levels, leading to promising risk reduction of shriveling chronic disease^[15].

 

Morinda citrifolia, commonly known as great morinda, Indian mulberry, nunaakai (Tamil Nadu, India), dog dumpling (Barbados), mengkudu (Indonesia and Malaysia), Kumudu (Balinese), pace (Javanese), beach mulberry, cheese fruit ^[16] or noni (from Hawaiian) is a tree in the coffee family, Rubiaceae. Morinda citrifolia's native range extends through Southeast Asia and Australasia, and the species is now cultivated throughout the tropics and widely naturalised.^[17] All parts of the plant have been used in traditional medicine.^[18] The fruit juice is high in demand in unconventional medicine for different kinds of illnesses such as arthritis, diabetes, cancer, AIDS, gastric ulcers, mental depression and poor digestion.^[19] With this background, the current study was designed to screen for the presence of phytochemicals and gauge the efficacy free radical scavenging activity in different parts of Malaysian Mengkudu with different solvent extracts.

 

MATERIALS AND METHODS

Plant materials

The leaves and fruits of Mengkudu were collected in Kuala Terengganu, Malaysia. They were later verified and authenticated at UniSZA Herbarium, Gong Badak Campus, Kuala Terengganu, Malaysia [Voucher no. 00217].

 

Chemicals

Major chemicals, buffers and reagents used in this study are Nacalaitesque, Kyoto - Japan, Gibco -Aukland, Calbiochem, Darmstadt - Germany, Sigma - USA. All the solvents used for extraction were analytical grade (purity > 99%) and were procured from Axon scientific Sdn Bhd., Selangor, Malaysia. 

 

Preparation of extracts for phytochemical analysis

The plant material (leaves and fruits) were air dried in the laboratory at room temperature. It was then powdered and was extracted with hot water by boiling for 30 minutes to get the aqueous extract. The extract obtained was concentrated and dried under controlled temperature (60^oC).The dried powder was successively extracted with other solvents and kept in an orbital shaker for overnight. Fresh fruit was homogenized with solvent and then extracted. The obtained extract was filtered with Whatman No. 42filter paper (125 mm) and the filtrate was collected and used for experimental analysis.

 

Phytochemical analysis

These studies were performed according to the standard methods^[20-22] for alkaloids, glycosides-cardio active aglycones, saponin glycosides, flavonoids, phenols, steroids, proteins and reducing sugars.

 

Tests for free radical scavenging assay

The antioxidant activity was evaluated using ethanol, chloroform and aqueous extracts for non-enzymatic antioxidants, ascorbic acid, β-carotene, tocopherol, phenol, protein with -SH group, reducing carbohydrate and sterol. Reducing power assay and super oxide radical scavenging assay was also performed with the above extracts.

 

Estimation of β–carotene (vitamin A) was done by the method of Carr& Price.^[23] Estimation of ascorbic acid was done by the method of Omaye.^[24] Estimation of tocopherol (vitamin E) was done by Rosenberg method.^[25] The reducing power of the extract was determined according to the method of Oyaizu.^[26] The superoxide radical scavenging ability was assessed by the method of Nishimik^.[27] Total phenol was determined using Lowry’s method.^[28] Sterol was estimated by Liebermann Burchard method.^[29]

 

Statistical analysis

Experimental results are expressed as means ± SD. All measurements were replicated ten times. The data was analyzed by an analysis of variance i.e. one way ANOVA and student ‘t’ test using Graph Pad Quick Calcs.

 

 

Figure 1.Level of β-carotenein aqueous, ethanol and chloroform extracts of MalaysianMengkudu per 100g

 

Fig. 2. Level of tocopherol in aqueous, ethanol and chloroform extracts of Malaysian Mengkudu per 100g

 

 

Table 1: Qualitative analysis of the phytochemicals of aqueous and organic extracts of Malaysian Mengkudu fruit.

Phytochemicals Analyzed	Mengkudu fruit
	Aqueous	Ethanol	Chloroform
Alkaloids	+++	++	=/=
Indole alkaloids	+++	+++	=/=
Lepac alkaloids	++	+	=/=
Cardio active aglycons	++	++	=/=
Saponin glycosides	++++	+++	=/=
Flavonoids	++	+	=/=
Phenols	++++	+++	=/=
Sterol	+++	++	+++
Proteins	+++	=/=	=/=
Carbohydrates	+++	=/=	=/=

+Presence of phytochemicals; ++Definite presence; +++Definite heavy presence;++++Definite heaviest presence; =/=Not done

RESULTS AND DISCUSSION:

The results of the phytochemical analysis were reported in Table 1. Table 1 shows the qualitative analysis results of the phytochemicals of aqueous and organic extracts of fruit of Mengkudu which includes the presence of alkaloids, indole alkaloids, lepac alkaloids, cardio active aglycons, saponin glycosides, flavonoids, phenols, sterol, proteins, and carbohydrates.

 

Figure 1 reveals that vitamin–A (β-carotene) content is highest in the leaf, high in dry fruit and less in fresh fruit. Chloroform extract has very high β-Carotenethan aqueous and ethanolic extract. β-carotene as a powerful, free radical scavenger (singlet oxygen) and chain breaking antioxidant.

 

 

Fig. 3 Level of ascorbic acid in aqueous, ethanol and chloroform extracts of Malaysian Mengkudu per 100g

 

Fig. 4 Level of polyphenol in aqueous, ethanol and chloroform extracts of Malaysian Mengkudu per 100g

 

Fig. 5 Level of protein in aqueous, ethanol and chloroform extracts of Malaysian Mengkudu per 100g

 

The function of β-carotene as a radical scavenging antioxidant can protect the cells from oxidative damage. Several clinical trials showed regression in precancerous lesions of the cervix and the lung as well as the oral cavity with the administration of β-carotene.^{[30, 31]}

 

In this study as depicted in figure 3, it was found that ascorbic acid (vitamin C) content is in the following order.

Dry Fruit> Fresh Fruit >Leaf;

Aqueous Extract >Ethanolic Extract > Chloroform Extract.

 

Ascorbic acid is an excellent hydrophilic antioxidant; it readily scavenges ROS and peroxyl radical and also acts as a co-antioxidant by regenerating the vitamin- A, E and GSH from radicals.^{[32, 33]}  Figure 2 indicates the high tocopherol content in fresh fruit with chloroform extract. Tocopherol is a fat soluble vitamin which can be extracted effectively by chloroform. Mengkudu has very high ascorbic acid along with tocopherol content. Vitamin C and E are synergistic antioxidants. Regeneration of vitamin E requires ascorbic acid, an aqueous phase antioxidant.

 

Figure 4 reveals that a high content of phenolic compounds was found in aqueous extract indicating that phenolic compounds in Mengkudu were mainly soluble in water. Phenolic compounds are likely to contribute to the radi­cal scavenging activity of extract. Polyphenols are a large class of compounds, occur naturally in food plants. The flavonoids are the largest and best studied group of these. Polyphenols are currently sold as dietary supplements and/or herbal remedies. They have antioxidant, antimuta­genic, anti-estrogenic, anti-carcinogenic and anti-inflam­matory effects that might potentially be beneficial in preventing disease. Epidemiologic findings revealed that polyphenols have cardio protective effects which include inhibition of platelet aggregation and vascular relaxation through the production of nitric oxide which decrease LDL oxidation and prevent atherosclerosis. ^{[33, 34]}

 

Figure 5 shows excellent protein content in ethanolic extract of Mengkudu. It may have more hydrophobic amino acids and lesser hydrophilic amino acids containing proteins. So they are extracted more in ethanol than in aqueous extract. Amino acids, peptides, such as carnosine and anserine, and proteins are common food components. Amino acids were found to be efficient antioxidants in model experiments. Their application is advantageous in mixtures with other inhibitors as they often act as synergists of phenolic antioxi­dants and as chelating agents. Amino acids convert hydro­peroxides into imines, and sulphur containing amino acids reduce hydroperoxides into the respective inactive hydrox­ylic derivatives. Methionine and selenomethionine were found to be more active than a-tocopherol in olive oil. ^[35]

 

Figure 6 shows that Mengkudu has high carbohydrates in aqueous extract indicating that carbohydrates in Mengkudu were soluble in water. These are likely to contribute to the radical scavenging activity and reducing power of the extract. Simple carbohydrates have antioxidant proper­ties. Hydroxyl radicals generated by a Fenton reaction induce damage on simple carbohydrates with a conse­quent free radical scavenging activity. Carbohydrate activi­ties were measured by different methods as spin-trapping of hydroxyl radical and electron paramagnetic resonance detection and 1, 1-diphenyl-2-picrylhydrazyl quenching. Carbohydrate damage was evaluated in a Fenton system by measuring the reactive substances to thiobarbituric acid, by their decreased detection with an HPLC test, and by a gas chromatographic determination of formic acid from sugar oxidation.

 

Figure 6. Level of reducing sugars in aqueous, ethanol and chloroform extracts of Malaysian Mengkudu per 100g

 

Different intensities of damage and scavenging were found according to molecular structure, and some hyphotheses on the carbohydrate action against free radicals were attempted. The assayed disaccharides were shown to be more active toward and less damaged by hydroxyl radical than monosaccharides. ^{[36, 37]}

 

Figure 7signposts that Malaysian Mengkuduleaf, fresh fruit and dry fruit have excellent sterol content both in organic and aqueous extracts. This may be due the presence of sterol which is extractable with organic and aqueous solvents. Mengkudu was found to have steroidal saponins and steroidal alkaloid which contributes total sterol content. Mengkudu has β-sitosterol- an important group of antioxidant. Plant sterols inhibit the intestinal absorption of cholesterol. Functional food ingredient derived from phytosterols has been clinically proven to have significantly lower Low Density Lipoprotein (LDL) or “bad” cholesterol when consumed in different foods. So they have a hypocholesterolemic action. They also inhibit endogenous synthesis of cholesterol by inhibiting and repressing the rate limiting enzyme Hydroxy methyl glutarylcoA (HMG-CoA) reductase in cholesterol synthesis. Steroidal glycosides in plant foods have estrogenic antiestrogenic actions and are known as phytoestrogen. These have antibacterial and antifungal actions.

 

Figure 7.Level of sterol in different extracts of MalaysianMengkuduin mg

 

Figure 8.Superoxide Radical Scavenging of Malaysian Mengkudu in%.

 

Figure 9.Reducing power of different extract of Malaysian Mengkuduat 700 nm.

 

 

They also produce typical estrogen responses with a biological activity 1/500 to1/1000 of estradial. Studies proved that phytoestrogens lower the incidence of steoporosis, breast and uterine cancer. ^[38]

 

Saponins present in Mengkuduare natural surfactants or detergents. It can be used as foaming agents for beverages. Saponins have a stypic activity which can be utilized in dissolving thrombus. Recent studies have suggested that the low serum cholesterol levels of Masai tribes in East Africa who consume a diet very high in animal products, cholesterol and saturated fat with saponin rich herbs. Saponins act by binding with bile acids and cholesterol. So it cleans or purges fatty compounds from the body, lowering the blood cholesterol level. Digitalis is a saponin used as heart tonic to strengthen contractions of the heart muscle. Saponins are active antifungal agents. ^[39]

 

The alkaloid present in Mengkudu is an indole alkaloid. The aromatic nitrogen heterocyclic is a potent cancer fighter/ anticancer agent, blocking carcinogenic substances before they reach their cellular targets and eliminating DNA damage in cell nuclei. It may also turn out to be an important chemical tool in fighting breast cancer because it inhibits estrogen induced growth of cancer cells and converts the more dangerous forms of estrogen to safer forms ^{[40, 41]}.

 

Isoflavones present in Mengkudu have a role similar to hormones, and act as phytoestrogens. They benefit humans in four ways as cancer enzyme inhibitor, as antioxidants and as immune system enhancers or stimulants. Phytoestrogens compete with estrogen for binding to estrogen receptors. Their use could have beneficial effects on preventing osteoporosis and sex hormone-mediated malignancy such as breast and prostate cancer. Clinical trials identified the potential efficacy of isoflavones in the prevention of coronary heart disease, osteoporosis, breast and prostate cancer ^{[42, 43]}.

 

Figure 8 specifies the outstanding superoxide radical scavenging ability found in almost all extracts of Mengkudu. Superoxide anions are one of the potent reactive oxygen species (ROS) which are produced from molecular oxygen due to oxidative enzymes^[44] of body as well as via non enzymatic reaction such as autoxidation by catecholamine ^[45]. In the present study superoxide radical reduces NBT to a blue colored for mazan that is measured at 560 nm^[46].

 

Figure 9 presents the exceptional reducing power of Mengkudu. In the reducing power (Fe³⁺- Fe²⁺ transformation ability) assay, the presence of antioxidants in the extracts would result in the reducing of Fe ³⁺ to Fe²⁺ by donatingan electron. Amount of Fe²⁺ complex can be then be monitored by measuring the formation of Perl's Prussian blue at 700 nm ^[47]. Antioxidant activity has been reported to be concomitant with development of reducing power. This shows that extracts might contain reductones, like ascorbic acid, reducing sugar, thiol group containing protein which could react with free radicals to stabilize and terminate radical chain reaction.

 

CONCLUSION:

These findings suggest that the promising antioxidant, cardio active (aglycones), astypic,  antimitotic, antimicrobial and antitumour properties of the Malaysian Mengkudu could be exploited against oxidative stress, cancer, ageing, Ischemic heart disease in dissolving thrombus, microbial infections and hormone replacement therapy (HRT) justifying their use in traditional medicine. This plant extract has assumed an increased importance in medicine and in health care industry and further work on the above suggested aspects may be given prominence. Phytochemicals occur naturally in plant foods which create an entirely new philosophy of “functional foods,” eating not just to sustain minimal basic health but also eating to prevent disease. In the future, we may tailor our diets to include the foods that will best address our personal health problems and risks as well as maintain optimal health.

 

ACKNOWLEDGEMENT:

This study was supported by the Program for Research, Universiti Sultan Zainal Abidin, Malaysia, vide UniSZA/R0172/12Apr’2012.

 

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