Evaluation of nutritional value and antioxidant activity of Leea macrophylla Roxb. ex Hornem. root and leaf

Abstract

Background:

Hanshia dabar or hathkan is one of the folklore plant of Odisha, the botanical source of which is Leea macrophylla Roxb. ex Hornem., family Vitaceae. Its root and leaves are eaten as vegetables, and used for Rasayana purpose (tonic and alterative properties).

Aim:

The aim is to assess the nutritive value and antioxidant potential of the root and leaves of L. macrophylla with compound leaf.

Materials and methods:

Nutritional parameters such as energy value, carbohydrate, protein, true protein, fat, mineral contents, and Vitamin A, Vitamin C of the root and leaves of the plant were evaluated with standard procedures. The in vitro antioxidant properties of the root and leaf of L. macrophylla were screened through 1,1diphenyl2picrylhydrazyl (DPPH) assay, the ferric reducing antioxidant power (FRAP) assay, and phosphomolybdenum assay.

Results:

Root is having higher energy value (391.87 Kcal/100 g) as compared to leaf (353.62 Kcal/100 g). L. macrophylla root and leaf showed the presence of carbohydrates (30.65% and 21.72%), protein (8.78% and 9.37%), true protein (6.85% and 7.23%), fat (0.77% and 1.89%), iron (723.80 ppm and 285.07 ppm), zinc (26.51 ppm and 13.75 ppm), manganese (44.88 ppm and 35.00 ppm), phosphorous (0.07 ppm and 0.03 ppm), calcium (7806.90 ppm and 3862.80 ppm), Vitamin A (3.63 mg/g and 2.47 mg/g), and Vitamin C (8.49 mg/g and 6.7 mg/g), respectively. Percentage scavenging of DPPH radical was found to rise with an increase in concentration. IC50 values of root and leaf, by DPPH assay, were 66.46 and 110.68 μg/ml, respectively. In the FRAP assay, the antioxidant activity of the methanolic extract of leaf (507.06 μmol/l) was found to be more than root (455.93 μmol/l). The total antioxidant capacity of root and leaf were 20.15 and 17.90 mg, equivalent to ascorbic acid on a dry weight basis, respectively.

Conclusion:

Root and leaf of L. macrophylla has the highest energy value, contains carbohydrate, protein, fat, iron, zinc, manganese, phosphorous, calcium, Vitamin A, and Vitamin C, and possesses antioxidant capacity.

Introduction

Daily intake of nutrition is essential for the growth and optimal functioning of the body. Food is consumed to compensate daily requirements of the body of macro- and micro-nutrients and as a source of energy. Antioxidants are natural substances that help in preventing or delaying some types of cell damage. Vegetables and fruits are rich sources of antioxidants that’s why their consumptionfound to be healthy.[1] These substances play a crucial role in a situation of oxidative stress in the body. Antioxidants prevent free radical-induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition.[2]

Traditional uses of Leea macrophylla Roxb. ex Hornem have been reported from two countries and nine states of India by 36 tribes/communities in 33 diseased conditions, used in Odisha in the name of Hanshin Dabar or Hatkhan,[3] which is used as a source drug of Hastikaranapalsha in Ayurveda.[4] Conventionally, vegetables are used as one of the major sources of food items. Root and leaves of L. macrophylla have been used as a vegetable since ancient times.[5,6,7,8]

L. macrophylla possess unifoliate, trifoliate, or 1–3 pinnate leaves.[9,10] The unifoliate variety of L. macrophylla has been reported for its antioxidant status.[11] Vitamins and minerals studies have been reported, but the author did not give a single clue regarding the variety of the plant, whether unifoliate or other else.[12,13] The present study was carried out to find out the nutritional richness and antioxidant status of the compound leaf variety of L. macrophylla.

Materials and methods

Collection and preservation of the sample

The plant L. macrophylla Roxb. ex Hornem. of family Vitaceae growing in Gandhamardan hill ranges, Paikmal, Odisha, was identified based on its morphological characteristics with the help of local taxonomists, botanists, botanical texts, and flora[9] [Figure 1] during September 2017 which was previously authenticated by BSI, Kolkata (Specimen No. CNH/2016/Tech. II/68 dated January 31, 2017) as L. macrophylla Roxb. ex Hornem., family-Vitaceae [Figure 2]. A specimen of the sample herbarium was deposited in the Pharmacognosy laboratory, IPGT and RA, Jamnagar, vide specimen no. Ph. M: 6234/2017 for further references [Figure 3]. The collected plant samples were shaken to remove adherent soil and dirt. The roots and leaves were separated from the stem and washed under running fresh water [Figures 4 and 5]. They were chopped separately, washed, shade dried, powdered, sieved through 80# mesh, and preserved in an airtight glass vessel for further study.

Figure 1.

Leea macrophylla in natural habitat

Figure 2.

Certificate from BSI

Figure 3.

Herbarium Ph.M: 6234/2017

Figure 4.

Root of Leea macrophylla

Figure 5.

Leaves of Leea macrophylla

Nutritional evaluation

Energy value (the calorific value) was estimated (in Kcal) by multiplying the percentage of crude protein, crude lipid, and carbohydrate by the recommended factor (2.44, 8.37, and 3.57, respectively) used in the analysis. The caloric value was determined based on the Atwater factor.[14]

Total carbohydrates were estimated by the phenolsulfuric acid method.[15] The crude protein was determined by the Kjeldahl method with slight modification, and absorbance was noted at 470 nm.[16] True protein content present in the samples was estimated by the FolinLowry method.[17] Estimation of fat was performed using n-Hexane as solvent by the Soxhlet extraction method.[16]

Vitamin A estimation was done using HPLC-Waters^™ 600 HPLC System and used (Mobile Phase: A-Methanol; B-0.02M H₃PO₄ (pH 3.54), standard preparation: 10 mg of the standard was dissolved in 1 ml of methanol.[18] Vitamin C was estimated by the method reported by Omaye et al., (1979[19] in which 2,4-dinitrophenyl hydrazine reagent was used and absorbance was read at 520 nm in a spectrophotometer.

Microwave plasmaatomic emission spectrometry (MP-AES 4200 Agilent) was used to determine iron, zinc, manganese, and calcium.[20] The Vanado-MolybdoPhosphuricacid colorimetric method[21] was used to determine the phosphorus content of acid extractions.

Antioxidant assay

1,1diphenyl2picrylhydrazyl (DPPH) radical scavenging activity was performed by the method described by Akter et al.[22] with slight modification. The ferric reducing antioxidant power (FRAP) was assessed according to Benzie and Strain[23] using a Hewlett-Packard 8453 diode array spectrophotometer. The total antioxidant capacity of the methanolic extract of samples was evaluated by the green phosphomolybdenum complex formation according to the previously described method of Prieto et al.[24] The absorbance of the mixture was measured at 695 nm.

Results and Discussion

Nutritional evaluation

Both root and leaf of L. macrophylla are a good source of energy and micronutrients. It possesses iron, zinc, phosphorous, manganese, and calcium along with protein, fat, and carbohydrate. It has the highest amount of energy content. The observed results are presented in detail in Table 1.

Table 1.

Nutritional value, mineral, and vitamin contents of Leea macrophylla root and leaves on a dry weight basis

Parameter	Results
	Root	Leaf
Nutritional value
Carbohydrate (%)	30.65	21.72
Total protein (%)	8.78	9.37
True protein (%)	6.85	7.23
Fat (%)	0.77	1.89
Energy value (kcal/100 g)	391.87	353.62
Mineral composition (ppm)
Iron	723.80	285.07
Zinc	26.51	13.75
Manganese	44.88	35.00
Phosphorus	0.07	0.03
Calcium	7806.90	3862.80
Vitamins (mg/g)
Vitamin A	3.63	2.47
Vitamin C	8.49	6.7

A slightly higher energy value is found in root (391.87 Kcal/100 g), followed by leaf (353.62 Kcal/100 g). [Table 1] That may be the reason that drugs root is commonly employed as a tonic and age sustainer.[5]

The root powder is found to be a good source of carbohydrates (30.65%), protein (8.78%), true protein (6.85%), and fat (0.77%). Estimation of mineral composition shows that root powder contains micronutrients such as iron (723.80 ppm), zinc (26.51 ppm), manganese (44.88 ppm), phosphorus (0.07 ppm), and calcium (7806.90 ppm). Vitamin A and Vitamin C are found in the quantity of 3.63 mg/g and 8.49 mg/g, respectively. [Table 1]

Leaf powder is found to be a good source of carbohydrates (21.72%), protein (9.37%), true protein (7.23%), and fat (1.89%). Leaf powder contains micronutrients such as iron (285.07 ppm), zinc (13.75 ppm), manganese (35.00 ppm), phosphorus (0.03 ppm), and calcium (3862.80 ppm). Vitamin A and Vitamin C are found in the quantity of 2.47 mg/g and 6.7 mg/g, respectively. [Table 1]

Except for total protein, true protein, and fat content, all other nutritional contents, minerals, vitamins, and energy value of root is higher than leaf of the plant.

Both root and leaf of the plant are used as a vegetable by the tribals[5] seems to be justifiable as these contains nutritional elements and minerals. Also it supports as being used as a tonic and age sustainer because of having good energy value.[5] Further, it is also found to be a good source of calcium and phosphorous, which helps in the management of bone fractures. The present study justifies its use in a bone fracture as maximum ethnomedicinal claims of the drug are in the management of bone fracture.[5]

Both root and leaf of the plant are good sources of macro- and micro-nutrients, vitamins, etc., Many nutritional components play a variety of roles in the wound healing process.[25] Suboptimal nutrition can alter immune function, collagen synthesis, and wound tensile strength, all of which are essential in the wound healing process.[25] Thereby supporting the plants ethnomedicinal claim of being used for wound healing purposes.[5]

Antioxidant assay

1,1diphenyl2picrylhydrazyl radical scavenging activity

The IC50 values of methanolic extract of root, leaf, and ascorbic acid were found to be 66.46 μg/ml, 110.68 μg/ml, and 109.16 μg/ml, respectively. [Table 2] Percentage scavenging of DPPH radical was found to rise with increasing concentration of the crude extract. [Graph 1]

Table 2.

Antioxidant values of root and leaf of Leea macrophylla

Samples	DPPH assay (IC50 values)	FRAP assay (µmol/L)	Phosphomolybdenum assay total antioxidant capacity (mg equivalent to ascorbic acid on a dry weight basis)
Ascorbic acid	109.16	-	-
Root of L. macrophylla*	66.46	455.93	20.15
Leaf of L. macrophylla*	110.68	507.06	17.90

*MeOH extract of root and leaf were used in all three methods studied. DPPH: 1,1diphenyl2picrylhydrazyl, FRAP: Ferric-reducing antioxidant power, L. macrophylla: Leea macrophylla

Graph 1.

1,1diphenyl2picrylhydrazyl % scavenging activity of standard ascorbic acid, root, and leaf samples of Leea macrophylla

As compared with standard ascorbic acid, the IC50 value in the DPPH assay of root is lower than standard, while of leaf is marginally higher, which clearly shows that the root of the plant possesses high antioxidant properties and the leaf has less antioxidant properties.[26] By the method adopted, the root sample showed higher antioxidant activity as compared to standard and leaf sample.

Ferric-reducing antioxidant power assay

The reducing potential of an antioxidant reacting with a ferric tripyridyltriazine (Fe³⁺-TPTZ) complex and producing a colored ferrous tripyridyltriazine (Fe²⁺-TPTZ) is measured by FRAP assay.[27] In general, the reducing properties of the substances are associated with the presence of various compounds which exert their action by breaking the free radical chain by donating a hydrogen atom.[28] In a redox-linked colorimetric reaction, the FRAP assay treats the antioxidants in the sample as a reductant.[29] In the present study, the trend for ferric ion reducing activities of all the test drugs is shown in Table 2 and Graph 2 and 3 depicts FeSo₄ standard concentration and FeSo₄ reducing property of standard Ascorbic acid respectively. The FRAP value of the root sample is 455.93 μmol/l, while the leaf sample is 507.06 μmol/l, which indicates the antioxidant potential of both the samples studied.

Graph 2.

FeSo4 standard concentration

Graph 3.

FeSo4 reducing property of standard ascorbic acid

Phosphomolybdenum assay

The phosphomolybdenum method is a method of studying antioxidant activity that is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and the formation of a green phosphate/Mo (V) complex with the maximal absorption at 695 nm. This assay has been routinely used to evaluate the antioxidant capacity of different extracts.[24] Graph 4 represents standard ascorbic acid in phosphomolybdenum assay. Methanol extracts of all the drug samples are used to determine their antioxidant capacities by the formation of green phosphomolybdenum complex. The formation of the complex was measured by the intensity of absorbance in extracts at a concentration of 100 μg/ml at 95°C. Being simple and independent of other antioxidant measurements commonly employed, the assay was extended to plant polyphenols. In the ranking of the antioxidant capacity obtained by this method, root of L. macrophylla showed higher phosphomolybdenum reduction (20.15 Mg Equivalent to ascorbic acid on a dry weight basis) followed by leaf of L. macrophylla (17.90 Mg Equivalent to ascorbic acid on a dry weight basis) [Table 2]. This may be explained by the fact that the transfer of electrons/hydrogen from antioxidants depends on the structure of the antioxidants.

Graph 4.

Graph of standard ascorbic acid in phosphomolybdenum assay

Root and leaf of the plant, studied by the above three methods, show the presence of antioxidant activity and thereby support their use as a vegetable by the tribal peoples.

Recent studies showed that root and leaf of the plant are rich in flavonoids, phenolic contents and tannins. Flavonoids are phenolic substances isolated from vascular plants that act as antioxidants.[30] Flavonoids have been shown to be highly effective scavengers of most oxidizing molecules and various free radicals.[31]

Phenolic compounds are able to act as antioxidants in a number of ways. The antioxidant capacity of phenolic compounds is attributed to their ability of chelating metal ions involved in the production of free radicals.[32] However, phenolic compounds can act as pro-oxidants by increasing their ability to form free radicals.[33] Phenolic structures often have the potential to powerfully interact with proteins because of their hydrophobic benzenoid rings and the hydrogen-bonding potential of the phenolic hydroxyl groups. The potential of inhibiting some enzymes involved in the radical generation, such as various cytochrome P450 isoforms, lipoxygenases, cyclooxygenase, and xanthine oxidase, gives phenolics the ability to act as antioxidants.[34,35] Phenolic compounds also act as antioxidanat agent because their hydroxyl groups confer scavenging ability.[36]

Tannins exert their antioxidant activity by scavenging free radicals, chelating trace metals, and by binding proteins with suppression of their enzymatic activity.[37] Yokozawa et al.[38] showed that the scavenging activity of tannins increases in the presence of an ortho-dihydroxy structure and with an increase in the number of galloyl groups and molecular weight: the hydroxyl groups are responsible for the chelating and radical scavenging properties of these compounds.

It is well known that many polyphenolic compounds, such as phenolic acids, flavonoids, and tannins, possess remarkable antioxidant activities. Therefore, the rich presence of flavonoids, phenolic compounds, and tannins in L. macrophylla root and leaf may be the major contributors for its antioxidant activity as these compounds act as natural antioxidants.[39] As root and leaf of plant possess antioxidant activity, it supports their use as a tonic and vegetable. The antioxidant activity of root supports its use as an age sustainer because antioxidant substances delay the aging process and extend lifespan.[40]

Conclusion

Root and leaves of L. macrophylla, family Vitaceae, with compound leaves, possess good energy value and are good sources of carbohydrates, proteins, fat, iron, zinc, manganese, phosphorous, calcium, Vitamin A and Vitamin C. The antioxidant capacity of the plant’s root and leaf can be ascribed to the presence of flavonoids, phenolic compounds, and tannins in it.

Financial support and sponsorship

Financial support by IPGT and RA, Gujarat Ayurveda University, Jamnagar-361008, India, and technical support by Junagadh Agricultural University, Junagadh-362001, India.

Conflicts of interest

There are no conflicts of interest.