**Review: Potential Antioxidants from Tropical Plants**

Lai Teng Ling1 and U.D. Palanisamy2 *1University of Malaya 2Monash University Sunway Campus Malaysia* 

#### **1. Introduction**

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#### **1.1 Background**

Higher plants have been utilised as important sources of medicinal drugs and health products since ancient days. Our ancestors practice the use of plants in their daily life as medicines. Therefore, the inherited knowledge about the traditional medicine is a key factor that could promote the development of modern drugs. Advances in modernisation and progress are expected to maximise the benefits of traditional herbal medicines for public health care (Chan, 2003). Investigations about plants have yielded amazing discoveries and development in modern medicine. Scientists carry out chemical investigation and purification of plants to get purified compounds which contribute to its medicinal properties.

The Malaysian rainforest is rich in diverse species of flora and fauna. Our forests store plenty of plant species which are important sources of traditional medicine. From about 10,000 species of higher plants and 2000 species of lower plants available in Peninsular Malaysia, approximately 16% are identified to be useful in medicines (Lattif et al., 1984). There are still a great number of unexplored plants that have high potential to be developed into medicines.

To date, a huge number of plants have been studied for their potential sources of antioxidants. Plants contain a wide variety of free radical scavenging molecules, such as polyphenols, dietary glutathione, vitamins and endogenous metabolites. These natural products make good antioxidants. Plant-derived antioxidants have been shown to function as singlet and triplet oxygen quenchers, peroxide decomposers, enzyme inhibitors and synergists (Larson, 1988). Studies have proven the correlation between the intake of fruits and vegetables and the morbidity and mortality from degenerative diseases (Rimm et al., 1996). It is not known which specific dietary constituents are responsible for this association, but antioxidants are assumed to be the major compounds that play an important role (Gey et al., 1991). Epidemiological studies that analyse the health implications of dietary components rely on the intake estimates in sample populations found in databases that list the component's content in commonly consumed foods (Pellegrini et al., 2003).

Besides dietary sources, antioxidants can be obtained in bulk from food processing industries and agricultural-by-products. The respective by-products are seeds, peels, bark, mill wastes and trimming wastes. In the citrus industries, industrial-by-products may

Review: Potential Antioxidants from Tropical Plants 65

plants which are potential sources of new antioxidants. A study was carried out in our laboratory to evaluate selected Malaysian plants for its free radical scavenging, inhibition in lipid peroxidation, phenolic and content. This was followed with the heavy metal and elements analysis and its cytotoxicity against several cell lines. The plants were chosen based on its use in traditional application within the region. The investigation is essential to establish the phenolic content of selected Malaysian plants, its capability as potential antioxidant and ensure its safety (Ling et al., 2010a). Figure 1 shows the cytotoxicity activity

Fig. 1. Cytotoxicity activity of selected Malaysian plants on 3T3 mouse fibroblast cell at the concentration of 100µg/mL. \* designates significant difference from cell alone (P< 0.05), \*\* designates a significant difference from cell alone, (P< 0.01), ## designates a significant

In our study, ethanolic and aqueous extracts of more than seventy Malaysian plants were screened using the DPPH assay. After the initial screen, thirteen plant extracts were selected with IC50 values lower than 5mg/mL and further tested with other free radical scavenging assays and for its ability to inhibit lipid peroxidation.. The extracts were also evaluated for its total phenolic content, heavy metal content and cytotoxicity. In general, the ethanolic extracts were observed to be far better free radical scavengers than the aqueous extracts. Some of the extracts were more potent (IC50 values) than the commercial grape seed antioxidant preparation (Agricultural Research Institute Speyer, Germany) and vitamin C. Generally, ethanolic extracts showed better activity in free radical scavenging assays and inhibition in lipid peroxidation compared to their aqueous extracts as well as higher than the commercial grape seed preparation. Most of the selected plant extracts showed hardly any heavy metal contamination in the powderised plants, some extracts even showed the presence of essential trace mineral. Majority of the plant extracts did not exhibit antiproliferative effects on cultured mouse fibroblast and breast cancer cell indicating that most of the plant extracts are not cytotoxic to the cell been studied. We also observed a positive correlation between the ethanolic extract, phenolic content and antioxidant activity. We can

of the selected Malaysian plants.

difference from grape seed (P<0.01).

account for up to 50% of the total fruit weight (Bocco et al., 1998). The utilisation of the byproducts in those industries is beneficial to both the economy and the environment. The industrial-by-products, like peel and seed, are proven to have high antioxidant level which is even higher than the flesh and other parts of the fruit with the presence of high polyphenol content. This is true for *Viburnum opulus* seed (Cam et al., 2007), peach peel (Chang et al., 2000), apple peel (He & Liu, 2007), mangosteen peel (Moongkarndi et al., 2004) and grape seed and skin (Rockenbach et al., 2011).

Numerous studies have been carried out on other potential agricultural-by-products such as trees. They serve as one of the cheapest available source of antioxidants. Among different parts of the plants, leaves receive special attention, e.g. Etlingera genus (Chan et al., 2011), Olea europaea (Silva et al., 2006), *Ligustrum vulgare* (Agati et al., 2009) and *Stevia rebaudiana* (Tadhani et al., 2007) ; bark from *Casuarina equisetifolia* (Zhang et al., 2010), *Acacia confusa* (Chang et al., 2001), *Populus tremuloides Michx* (Diouf et al., 2009); root of *Medicago sativa* (Dalton et al., 1998) and *Carissa spinarum* (Hegde & Joshi, 2010) were also reported to contain antioxidants.

Despite the utilisation of agricultural-by-products for human consumption, the safeties of the products have raised much attention. Dietary exposure to heavy metals, especially cadmium (Cd), lead (Pb), zinc (Zn) and copper (Cu), has been identified as a risk to human health. Heavy metals may be present in trace amounts occurring naturally in plants grown in the soil (Boruvka et al., 1997). Heavy metals have also been found in herbal medicines from Malaysia, e.g. *Eurycoma longifolia* products (Ang et al., 2003). Studies in Malaysia showed that only 92% of the products complied with the quality requirement for traditional medicines in the country, however, they cannot be assumed to be safe from lead contamination because of batch-to-batch inconsistency (Ang et al., 2003). Cadmium is reported to accumulate in the kidney. There is overwhelming evidence that the cadmium induced tubular damage which is irreversible (Järup et al., 1998). Therefore, it is important to ensure that plants that are consumed by humans do not contain heavy metals higher than the permissible levels. This has to be monitored carefully to ensure the safety of plant parts used as nutraceuticals.

Generally, antioxidants extracted from plants show prooxidant activity at low concentration and antioxidant activity at higher concentrations (Yen et al., 1997). However, the opposite effect was observed in the case of ascorbic acid in the presence of transition irons (Halliwell, 1996). These findings remind us the importance of quantifying the prooxidant capacity of an extract that exerts high antioxidant activity and to interpret net antioxidant potential.

To date, there is no information concerning the profile of Malaysian plants, its antioxidant/prooxidant activity, cytotoxicity, heavy metal contamination and method of standardisation. The purpose of this study is to bridge this gap of knowledge. Direct beneficiaries of this research would be the general public, the herbal industries and the natural product researchers in Malaysia and elsewhere.

#### **2. Assessment of antioxidant capacity and cytotoxicity of selected malaysian plants**

Malaysia is rich in its biodiversity with over 12,000 flowering plant species, many of which are currently being used in traditional medicine. To date, a huge number of plants have been studied for its potential source of antioxidants. However, there exist no reports on the antioxidant activity, heavy metal and elemental analysis and cytotoxicity of Malaysian

account for up to 50% of the total fruit weight (Bocco et al., 1998). The utilisation of the byproducts in those industries is beneficial to both the economy and the environment. The industrial-by-products, like peel and seed, are proven to have high antioxidant level which is even higher than the flesh and other parts of the fruit with the presence of high polyphenol content. This is true for *Viburnum opulus* seed (Cam et al., 2007), peach peel (Chang et al., 2000), apple peel (He & Liu, 2007), mangosteen peel (Moongkarndi et al., 2004)

Numerous studies have been carried out on other potential agricultural-by-products such as trees. They serve as one of the cheapest available source of antioxidants. Among different parts of the plants, leaves receive special attention, e.g. Etlingera genus (Chan et al., 2011), Olea europaea (Silva et al., 2006), *Ligustrum vulgare* (Agati et al., 2009) and *Stevia rebaudiana* (Tadhani et al., 2007) ; bark from *Casuarina equisetifolia* (Zhang et al., 2010), *Acacia confusa* (Chang et al., 2001), *Populus tremuloides Michx* (Diouf et al., 2009); root of *Medicago sativa* (Dalton et al., 1998)

Despite the utilisation of agricultural-by-products for human consumption, the safeties of the products have raised much attention. Dietary exposure to heavy metals, especially cadmium (Cd), lead (Pb), zinc (Zn) and copper (Cu), has been identified as a risk to human health. Heavy metals may be present in trace amounts occurring naturally in plants grown in the soil (Boruvka et al., 1997). Heavy metals have also been found in herbal medicines from Malaysia, e.g. *Eurycoma longifolia* products (Ang et al., 2003). Studies in Malaysia showed that only 92% of the products complied with the quality requirement for traditional medicines in the country, however, they cannot be assumed to be safe from lead contamination because of batch-to-batch inconsistency (Ang et al., 2003). Cadmium is reported to accumulate in the kidney. There is overwhelming evidence that the cadmium induced tubular damage which is irreversible (Järup et al., 1998). Therefore, it is important to ensure that plants that are consumed by humans do not contain heavy metals higher than the permissible levels. This has to be monitored carefully to ensure the safety of plant parts

Generally, antioxidants extracted from plants show prooxidant activity at low concentration and antioxidant activity at higher concentrations (Yen et al., 1997). However, the opposite effect was observed in the case of ascorbic acid in the presence of transition irons (Halliwell, 1996). These findings remind us the importance of quantifying the prooxidant capacity of an

To date, there is no information concerning the profile of Malaysian plants, its antioxidant/prooxidant activity, cytotoxicity, heavy metal contamination and method of standardisation. The purpose of this study is to bridge this gap of knowledge. Direct beneficiaries of this research would be the general public, the herbal industries and the

**2. Assessment of antioxidant capacity and cytotoxicity of selected malaysian** 

Malaysia is rich in its biodiversity with over 12,000 flowering plant species, many of which are currently being used in traditional medicine. To date, a huge number of plants have been studied for its potential source of antioxidants. However, there exist no reports on the antioxidant activity, heavy metal and elemental analysis and cytotoxicity of Malaysian

extract that exerts high antioxidant activity and to interpret net antioxidant potential.

natural product researchers in Malaysia and elsewhere.

and *Carissa spinarum* (Hegde & Joshi, 2010) were also reported to contain antioxidants.

and grape seed and skin (Rockenbach et al., 2011).

used as nutraceuticals.

**plants** 

plants which are potential sources of new antioxidants. A study was carried out in our laboratory to evaluate selected Malaysian plants for its free radical scavenging, inhibition in lipid peroxidation, phenolic and content. This was followed with the heavy metal and elements analysis and its cytotoxicity against several cell lines. The plants were chosen based on its use in traditional application within the region. The investigation is essential to establish the phenolic content of selected Malaysian plants, its capability as potential antioxidant and ensure its safety (Ling et al., 2010a). Figure 1 shows the cytotoxicity activity of the selected Malaysian plants.

Fig. 1. Cytotoxicity activity of selected Malaysian plants on 3T3 mouse fibroblast cell at the concentration of 100µg/mL. \* designates significant difference from cell alone (P< 0.05), \*\* designates a significant difference from cell alone, (P< 0.01), ## designates a significant difference from grape seed (P<0.01).

In our study, ethanolic and aqueous extracts of more than seventy Malaysian plants were screened using the DPPH assay. After the initial screen, thirteen plant extracts were selected with IC50 values lower than 5mg/mL and further tested with other free radical scavenging assays and for its ability to inhibit lipid peroxidation.. The extracts were also evaluated for its total phenolic content, heavy metal content and cytotoxicity. In general, the ethanolic extracts were observed to be far better free radical scavengers than the aqueous extracts. Some of the extracts were more potent (IC50 values) than the commercial grape seed antioxidant preparation (Agricultural Research Institute Speyer, Germany) and vitamin C. Generally, ethanolic extracts showed better activity in free radical scavenging assays and inhibition in lipid peroxidation compared to their aqueous extracts as well as higher than the commercial grape seed preparation. Most of the selected plant extracts showed hardly any heavy metal contamination in the powderised plants, some extracts even showed the presence of essential trace mineral. Majority of the plant extracts did not exhibit antiproliferative effects on cultured mouse fibroblast and breast cancer cell indicating that most of the plant extracts are not cytotoxic to the cell been studied. We also observed a positive correlation between the ethanolic extract, phenolic content and antioxidant activity. We can

Review: Potential Antioxidants from Tropical Plants 67

*Azadirachta indica* leaf 0.74±0.46 0.50±0.02 0.91±0.58 *Mangifera indica* leaf 0.17±0.02 0.22±0.03 1.32±0.29 *Garcinia mangostana* peel 0.11±0.02 0.17±0.04 1.62±0.40 *Nephelium lappaceum* **peel 0.12±0.05 0.05±0.04 0.48±0.37**  *Psidium guajava* leaf 0.18±0.08 0.20±0.03 1.27±0.60 *Fragaria x ananassa* **leaf 1.87±0.80 0.99±0.34 0.64±0.45**  *Lawsonia inermis* **leaf 1.3±0.18 0.54±0.07 0.43±0.11**  *Syzygium aqueum* **leaf 0.22±0.02 0.13±0.03 0.62±0.13**  *Nephelium lappaceum* leaf 0.33±0.03 0.76±0.42 2.37±1.40 *Peltophorum pterocarpum* leaf 0.17±0.12 0.11±0.03 0.83±0.44 *Peltophorum pterocarpum* bark 0.10±0.04 0.08±0.03 0.94±0.66 *Artocarpus champeden* leaf 0.30±0.21 0.75±0.51 3.82±4.24 *Nephelium mutobile* leaf 0.24±0.03 0.18±0.06 0.76±0.28 *Vitis vinifera* **seed 0.15±0.10 0.07±0.02 0.59±0.33**  Table 1. DPPH scavenging activity, Pro-oxidant activity and ProAntidex in ethanolic extracts of selected Malaysian plants and standard. ProAntidex was devised using the ratio of prooxidant activities to the IC50 DPPH scavenging activity. All values represent means ± SD, *n*=3.

 **Extract Part (IC50, mg/ml) (mg/ml)** 

**Ethanolic Plant DPPH Pro-oxidant Pro-Antidex** 

**Aqueous Plant DPPH Pro-oxidant Pro-Antidex** 

*Azadirachta indica* leaf 0.96±0.14 1.13±0.01 1.20±0.18 *Mangifera indica* leaf 0.49±0.39 1.03±0.88 2.03±1.38 *Garcinia mangostana* peel 1.66±2.4 3.04±0.08 7.26±6.12\*\* *Nephelium lappaceum* peel 0.54±0.15 0.55±0.37 1.08±0.65 *Psidium guajava* leaf 0.22±0.01 0.42±0.33 1.89±1.43 *Fragaria x ananassa* leaf 0.37±0.07 0.58±0.003 1.60±0.29 *Lawsonia inermis* **leaf 3.71±0.34 1.41±0.21 0.38±0.06**  *Syzygium aqueum* leaf 0.33±0.07 0.26±0.09 0.88±0.51 *Nephelium lappaceum* leaf 0.67±0.02 >2 NA *Peltophorum pterocarpum* leaf 0.16±0.05 0.17±0.01 1.14±0.45 *Peltophorum pterocarpum* bark 0.20±0.12 0.23±0.01 1.48±0.88 *Artocarpus champeden* leaf 0.22±0.01 0.20±0.001 0.93±0.05 *Nephelium mutobile* **leaf 3.76±0.27 0.18±0.01 0.05±0.01**  *Vitis vinifera* **seed 0.46±0.18 0.24±0.11 0.59±0.35**  *Green tea* NA 0.28±0.04 0.23±0.07 0.82±0.28 Emblica™ NA 0.31±0.07 0.27±0.12 0.69±0.18 Vitamin C NA 0.01±0.00 0.03±0.35 4.10±3.36 Table 2. DPPH scavenging activity, pro-oxidant and ProAntidex in aqueous extracts of selected Malaysian plants and standards. ProAntidex was devised using the ratio of prooxidant activities to the IC50 DPPH scavenging activity. All values represent means ± SD,

 **Extract Part (IC50, mg/ml) (mg/ml)** 

*n*=3. \*\*Designates a significance difference from EmblicaTM, *p*<0.01.

conclude that although a broaden use of these plants are in aqueous form, its commercial preparation can be achieved using ethanol since a high total phenolic content and antioxidant activity was seen in this preparation. It is desirable that these extracts be further purified to gain a better understanding of the active compounds contributing to its antioxidant activity.

Similar results were observed in the lipid peroxidation inhibition studies (Palanisamy et al., 2008). There was a strong correlation between antioxidant activity and the total phenolic content of the extracts. The high antioxidant extracts had below the permissible value of heavy metal content for nutraceutical application. Most of the extracts were also not cytotoxic to 3T3 and 4T1 cells at concentration as high as 100μg/mL (Ling et al., 2010a).
