Isolation and Characterization of Some Phytochemicals from Indian Traditional Plants

The present study was designed to evaluate relative contribution of different polyphenols (total phenolics, flavonoids, flavonols) and their antioxidants activities in aqueous extracts of different parts of some plants; Argemone mexicana, Datura metel, Calotropis procera, Thevetia peruviana, and Cannabis sativa. The antioxidants (total phenolics, flavonoids, flavones) were determined by chemical methods. The antioxidant capacities of these extracts were evaluated by FRAP assay. The results demonstrated that phenolic content was maximally present in leaves of T. peruviana. This plant exhibited minimum phenolic content in its flower as compared to other plants. The flower of D. metel contained maximum phenolic content. The flavonoids were present in highest quantity in leaves of C. procera while T. peruviana flowers showed maximum flavonoid content. The fruits of C. sativa contained maximum quantity of flavonoid as compared to other plants tested. The flower extract of C. sativa possessed highest FRAP value followed by A. mexicana and fruit of C. procera. The values of ratios of different polyphenolic compounds present in plant extracts indicated that flower of D. metel contained maximum total flavonoids and minimum phenolics. These results suggested that levels of total phenolics, flavonoids and their FRAP indices exhibited specificity to different plants and their parts.


Introduction
The extraction of plant constituents is essential to isolate biologically active compounds and in understanding their role in disease prevention and treatment and in knowing their toxic effects as well. However, meager information is available about the medicinal and pharmacological properties and biological activities of phytochemicals derived from some plants (Calotropis procera, Datura metal, Cannabis sativa, Argemone Mexicana, and Thevetia peruviana) commonly known to have toxic, narcotic and ornamental properties. The information available on these important plants indicates that not much attention has been paid towards studying their physicochemical properties as well as biological activities towards their potentials as antioxidants. Keeping this information in view, an endeavour has been made in this communication to determine some biochemical constituents and their properties into the aqueous extracts of aforesaid medicinally important plants commonly available in the northern part of India.
Calotropis procera, known as apple of Sodom or mudar, belongs to Apocynaceae family and is found in many countries such as Africa and Western and South Asia, as well as Indochina. It is known for its medicinal and pharmacological properties [1]. The milky sap of this plant is known to contain three toxic glycosides: (i) calotropin, (ii) uscharin, and (iii) calotoxin as well as steroidal heart poisons, known as cardiac aglycones [2]. The crude extract of this plant and its protein fraction possess high fibrinolytic and anticoagulant activity in rabbit and human plasma [3]. Aqueous extracts of different parts of this plants are shown to exert mild diuretic and cardiac as well as respiratory stimulating effects in experimental animals [4].
Datura metal, a well-known traditional Indian plant, is found throughout the warmer parts of the world and contains both the ornamental and medicinal properties. All parts of Datura plants contain high levels of tropane alkaloids, which are highly toxic to humans and other animals. This plant is known to possess analgesic [5,6], antioxidant, and antimicrobial properties [7]. dinitrogen trioxide (N 2 O 3 ), nitroxyl anion (NO − ), nitrous acid (HNO 2 ), and nitryl chloride (NO 2 Cl) [28,29].
There are evidences which suggest that by quenching the free radicals, antioxidants help reduce the risk of chronic diseases. These antioxidants are either endogenous (internally synthesized) or exogenous (consumed). Nowadays, the application of plant based antioxidants or natural antioxidants is replacing synthetic molecules because of toxicities associated with the later [30,31]. The phytochemicals like phenolic acids, polyphenols, flavonoids, flavonols, terpenoids vitamin C, vitamin E, carotenes, phenolic acids, phytate, and phytoestrogens scavenge the free radicals activity thus inhibiting the oxidative mechanisms that lead to emergence of various diseases as these molecules are electron rich. They donate electrons to ROS and neutralize these chemical species [32,33].
In this study we have selected the aforesaid five plants, namely, Calotropis procera, Datura metal, Cannabis sativa, Argemone Mexicana, and Thevetia peruviana known for their varied properties. C. procera has the medicinal and toxic constituents, D. metal contains narcotics and toxic substances, C. sativa possesses some narcotics, A. mexicana causes dropsy, and T. peruviana has medicinal and ornamental applications. In our laboratory, some of the properties of the phytochemicals present in aqueous extracts of different parts (leaves, stem, flowers, and fruits) of these plants have been explored. In this paper, the presence of polyphenolic contents (total phenolics, flavonoids, and flavonols) in these aqueous extracts as well as their antioxidant potentials have been demonstrated.

Preparation of Plant Extracts.
The fresh plant parts were collected washed with tap water followed by distilled water. 5.0 g of each was cut into several small pieces, minced well in a pestle and a mortar, and extracted with 50 mL of 50 mM Tris-HCl buffer at (pH 7.0). Freezing and thawing are done twice at the intervals of 2 h each followed by mechanical jerk by grinding in the pestle mortar in order to rupture the plant cell wall. The 10% (w/v) homogenate of each of the plant materials was prepared at 4-6 • C. The homogenate was filtered using Whatman's filter paper type 1. The volume of the filtrate was recorded. The filtrate was centrifuged at 1000 xg for 10 min under cooling (4-6 • C) conditions. The clear supernatant was used to estimate their antioxidant potential. The difference of the weights of the starting material and the residues was considered as the amount of the plant present in the extract.

Determination of Total
Phenolics. Folin-Ciocalteu method as described elsewhere [34] was employed for estimation of total phenolics in the aqueous plant extracts. An aliquot (100 µL) of the extracts was mixed with 2.5 mL Folin-Ciocalteu reagent (previously diluted with water; 1 : 10 v/v) and 2 mL (75 g/L) of sodium carbonate. The tubes were vortexed for 15 s and allowed to stand for 30 min at 40 • C for color development. The optical absorbance was recorded against reagent blank at 765 nm wavelength using the Thermoscientific Spectrascan UV2700 double beam spectrophotometer. The concentration of each plant extract was 0.1 g/mL. Total phenolic contents were expressed as mg/g n-propyl gallate equivalent.

Determination of Total Flavonoids.
Determination of total flavonoid content was done using the method already described elsewhere [35]. In brief, a volume of 0.5 mL of 2% AlCl 3 in ethanol solution was added to 0.5 mL of plant extracts. After 1 h incubation at room temperature, the absorbance was measured at 420 nm. Appearance of yellow color indicated the presence of flavonoids. The extract samples were evaluated at a final concentration of 0.1 g/mL. Total flavonoid contents were calculated as quercetin equivalent (mg/g).

Determination of Total Flavonols.
Total flavonols in the plant extracts were estimated by a known method described elsewhere [36]. In brief, 1.0 mL of 2% AlCl 3 in ethanol and 1.5 mL sodium acetate (50 g/L) solutions were added in 0.10 mL of extract solution. The absorption at 440 nm was monitored after 2.5 h of incubation at 20 • C. The sample extracts were evaluated at a final concentration of 0.1 mg/mL. Total flavonoid content was calculated as quercetin equivalent (mg/g).

Total Antioxidant Activity Determination by Ferric
Reducing Antioxidant Power (FRAP) Assay. The method as described by Benzie and Strain [37] with some modifications was employed for the estimation of antioxidant activity by FRAP assay. The stock solutions included 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl, and 1 mM FeCl 3 · 6H 2 O solution. TPTZ was dissolved in 40 mM HCl at 50 • C in water bath for 30-40 min till it completely dissolves. The fresh working solution was prepared by mixing 10 : 1 : 1 of acetate buffer, TPTZ, and FeCl 3 · 6H 2 O, respectively. The temperature of the working solution was maintained to 37 • C before starting the reaction by adding the plant extracts (100 µL) to 2 mL of the FRAP solution. The reaction mixture was incubated for 30 min in the dark condition. The optical absorbance of the colored product (ferrous tripyridyltriazine complex) was recorded at 593 nm. The standard curve was linear between

The Evaluation of Phenolics in the Aqueous Extracts of Different Parts of the Plants.
The data obtained after analysis of total phenolics as shown in Figure 1 was largely variable not only among the plants but also among their various parts. In case of A. mexicana, the highest phenolic content was found in flowers (14 mg/g) followed by leaf, fruit, and stem, with the values being 7.5, 4.62, and 2.5 mg/g, respectively. The aqueous extracts of D. metal showed a similar pattern with maximum phenolic content present in flowers (19.75 mg/gm) followed by leaf, fruit, and stem, with the values being 11, 5.5, and 1.25 mg/g, respectively. In C. procera, maximum phenolic content was present in leaves (14 mg/g). Other parts of C. procera such as fruit, flower, and stem, had values of 7.7, 6.7, and 2.7 mg/g, respectively.
Among the different parts of T. peruviana, maximum phenolic content was found in leaves (41 mg/g) followed by fruit, stem, and flower, with the values being 9.75, 7.3, and 5.75 mg/g, respectively. The flower of C. sativa contained maximum phenolic content (13.5 mg/g) followed by leaf and stem, with the values being 9.62 and 5.7 mg/g, respectively ( Figure 1). Upon comparison of all the five plants, the leaves of T. peruviana were found to have maximum phenolic content followed by leaves of C. procera, D. metal, C. sativa, and A. mexicana. Stems of all the plants did not contain a significant quantity of phenolics ( Figure 1). In case of flowers, D. metal contained maximum phenolic content followed by that of A.

The Evaluation of Ferric Reducing Antioxidant Power (FRAP) in the Aqueous Extracts of Different Parts of the Plants.
When these plant extracts were subjected to FRAP assay, flowers of C. sativa showed a significant antioxidant potential (74.8 ± 1.93 µM Fe ++ g −1 ) among all the plants. A. mexicana flowers, D. metal leaves, fruits from C. procera, and T. peruviana showed maximum antioxidant capacity, with the values being 69.1 ± 0.28, 24.7 ± 1.13, 41.3 ± 1.20, and 24.2 ± 0.31 µM Fe ++ g −1 , respectively (Table 1).

Discussion
It is well known that plant polyphenols, the secondary metabolites, are widely distributed in the plant kingdom and that they are sometimes present in surprisingly high concentrations [38]. Phenolic compounds are characterized by the presence of several phenol groups. By donating a hydrogen atom or an electron they make them very reactive in neutralizing free radicals, chelating metal ions in aqueous solutions [39]. The results of present study indicated that the amount of phenolic contents varied not only plantwise but also from one part of the plant to another.   [42]. These compounds act as free radical scavengers and thus help protect cells from oxidative toxicity [43][44][45]. Some workers have demonstrated the presence of phenolic compounds in the aerial parts of the plants including C. procera, T. peruviana, and C. sativa [46][47][48] but analysis of these phytoconstituents in the aqueous extracts of different specific parts of these plants has not been worked out.
It is reported that in the Fenton reaction, flavonoids as antioxidants interfere with the biochemical pathways which are involved in the generation of free radicals (ROS), quench them, chelate the transition metals, and make them redox inactive [49]. Commonly flavonoids occur as glycosides in plants and are considered to be very efficient as antioxidants. With different degrees of hydroxylation, oxidation, and substitution, the flavonoids have common diphenylpropane structure (C 6 C 3 C 6 ) [40,50].
The results of the present study reflected that the quantity of flavonoids varies from one plant to another and also into different parts of the plants. Maximum flavonoid content was present in the leaves of C. procera and flowers as well as fruits of T. peruviana when compared to D. metal, C. sativa, A. Mexicana, and T. peruviana. The stem of these plants contained low amount of flavonoid. Liu et al. have reported flavonoids content to be absent in Cannabis fruit [41]. Very recently, the levels of flavonoids in different medicinal plants have been reported by de Queiroz Siqueira et al. [51] and they have demonstrated the similar distribution pattern of the flavonoids in specific parts of different plants. According to a hypothesis proposed by Tattini et al. [52], flavonoids have protective functions during drought. Ryan et al. [53] have demonstrated that these molecules impart photoprotection. Likewise, Barceló and Poschenrieder [54] have shown that flavonoids in plants may help ameliorate toxicity of aluminium as they grow in soils contaminated with this heavy metal. Thus in addition to acting as antioxidants, flavonoids are also involved in the regulation of various physiochemical behaviours of plants.
The antioxidant capacity of the plant extract largely depends on both the composition of the extract and the test system [55]. The FRAP assay [37,56] measures antioxidant power with the help of an oxidant, that is, Fe 3+ . Reduction of ferric to ferrous ion at low pH produces a coloured ferrous-tripyridyltriazine complex. In the FRAP assay, reductants (antioxidants) present in the sample reduce the Fe (III)/tripyridyltriazine complex to the blue ferrous form. The change in absorbance and FRAP value of the antioxidants is proportional to each other [56]. The FRAP assay in spite of being simple and inexpensive does have few drawbacks too like the antioxidant capacity of certain antioxidants cannot be measured accurately by this assay such as iron (II) and SH group-containing antioxidants [37,[56][57][58].
The evaluation of FRAP value has been made in the aqueous extracts of different parts of the five plants tested in the present study and the results suggested that flowers of A. mexicana and C. sativa, D. metal leaves, and fruits from C. procera and T. peruviana exhibited maximum antioxidant capacity. The trend of FRAP values obtained from different plant parts having maximum antioxidant potential for being used in various pharmacological preparations is flowers of C. sativa > flowers of A. mexicana> fruits of C. procera > leaves of D. metal > fruits of T. peruviana. These results are in agreement with those reported by Katalinic et al. [42]. In another species of Datura, that is, Datura stramonium, Oseni [61], which could be attributed to substantial differences in the solubility of phytochemicals extracted into organic and aqueous solvents. The strong correlation observed in the present study between antioxidant activity, phenolics, and flavonoid content of different plants suggests a possible use of their partsin making the active ingredients of antioxidant supplement after removing their toxic ingredients, if any.

Conclusion
The results from the present study demonstrated that the leaves of T. peruviana contained the presence of the maximum phenolic content. This plant exhibited minimum phenolic content in its flower as compared to others. Phenolic contents were maximum in the flowers of D. metal. The flavonoids were present in highest quantity in the leaves of C. procera while the T. peruviana flowers showed maximum flavonoid content. The fruits of C. sativa contained maximum quantity of flavonoid as compared to other plants tested. The aqueous extract of the flower of C. sativa possessed highest FRAP value followed by the flower of A. mexicana and the fruit of C. procera. The values of ratios of different polyphenolic compounds present in the plant extracts indicated that the flower of D. metal contained maximum flavonoids and minimum phenolics. These results suggested that the levels of total phenolics and flavonoids contents as well their FRAP indices varied not only from one plant to the other but also in their different parts tested. These results indicated that despite the presence of some toxic ingredients, these plants contained high antioxidant activity and sufficient quantity of flavonoids and phenolics in their varying parts, which may be exploited for certain medicinal or pharmacological formulations.