Phytochemical Analysis of Selected Medicinal Plants from Eastern Ghats of Andhra Pradesh

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Free radicals are fragments of molecules with a very short half-life, generated through internal/ external sources, are highly reactive and damage macromolecules like proteins, DNA and lipids of cell membranes of living organisms.The most common reactive oxygen species are hydroxyl (OH), hydrogen peroxide (H2O2), super oxide anion (O2), and peroxyl radicals (ROO) and nitrogen derived free radicals are nitric oxide (NO), peroxyl nitrite anion (ONOO), Nitrogen dioxide (NO2) and Di-nitrogen trioxide (N2O3).The chemical constituents that retard or suppress oxidation or prolong the life of the oxidizable molecules/ inhibit the oxidation process are called antioxidants.Antioxidants from natural/ plant sources enhance the endogenous enzymes' anti-oxidative capability and reduce the risk of many free radical mediated diseases.Traditional medicinal systems use medicinal plants containing various chemical components such as alkaloids, polyphenols, glycosides and terpenoids, which showed pharmacological properties such as antioxidant and antimicrobial activities 1,2 .Of the estimated 2.5 to 5 lakhs plant species; very few have been screened for its biological or pharmacological activities 3 .In this connection, we selected the following water extract of six medicinal plants to screen phytochemical analysis and antioxidant potential.
Acalypha alnifolia Klein ex Willd., is a rare medicinal plant found in the forests of South India 4 .Acalypha belongs to the family Euphorbiaceae and is the fourth largest genus with about 450 species 5,6 .In traditional medicine, the plant has been used to treat dysentery, diabetes and as mosquito repellent 4,7,8 .
Caesalpinia bonduc Roxb.(family: Caesalpiniaceae) is locally known as Lata Karanja, is a prickly woody liana distributed throughout the hotter parts of India and Sri Lanka 9 It is a valuable medicinal plant and its different parts, such as bark, leaves, roots and seeds are utilized in traditional system of medicine.The roots are effective as an antiperiodic and antispasmodic properties 10 , the bark is a good remedy as anthelmintic and febrifuge and the leaves were reported as an emmenagogue 11 .The seeds are reported to have various pharmacological actions like antipyretic, antiperiodic, asthmatic and febrifuge 12,13 .
Carissa spinarum L. is a spinous evergreen shrub that belongs to the family Apocynaceae, distributed throughout dry localities in India.The plant has been used in the Ayurvedic medical system to treat liver problems, epileptic disease, microbial infections, cytotoxic, viral diseases, inflammation, arthritis and cancer 14 .The ripened fruits are edible and reported to have cardiac protective properties.
Commiphora caudata (Arn) Engl.belongs to the family Burseraceae, is a thorny shrub to medium sized tree distributed in dry deciduous forests of Peninsular India of Andhra Pradesh, Karnataka and Tamil Nadu 15,16 .It is commonly known as a hill mango because of the mango flavour of its stem bark.Traditionally, the bark has been used to treat diabetes, arthritis and obesity 17,18 .Fruits have been used to prepare pickles and gum is used as incense.
Moringa concanensis Nimmo, is a rare medicinal plant of the family Moringaceae.It is found in dry localities in Konkan, Andhra Pradesh.In Telugu is called Konda munaga/ Karumunaga, because of its similar morphological characters of drumstick plant M. oleifera 19 .Different parts of this plant have been reported to treat different human ailments such as leaves for gynic problems, hyper-tention, constipation, jaundice, skin cancers, diabetes and splenomegaly, stem bark for abortion and fruits for rheumatism, nervous disorders, curing liver and spleen diseases, gum for dental problems and flowers for aphrodisiac, leucorrhea and abortion 20 .
Terminalia tomentosa is a deciduous tree that belongs to the family Combretaceae.Generally, T. tomentosa is called a "crocodile bark tree" because the bark of this has characteristics feature like the skin of crocodile.It is found in Southern parts of the Indian subcontinent and other Southeast Asian countries 21 .In Ayurveda, the bark treats rheumatism, fever, urinary diseases and diabetes, vertigo, piles, constipation and chronic dysentery 22 .

Preparation of water extracts
The plant parts were cleaned with tap water, sliced and shade dried.The mixer grinder was used to grind the dried material to fine powder.The powdered material was mixed with double distilled water and boiled for 30 min and filtered.The filtrate was concentrated on water both.The phytochemical and antioxidant activities of crude water extract (WE) were analyzed.

Preliminary Phytochemical screening
Qualitative phytochemical screening of the selected six medicinal plant extracts was analyzed using standard methods 25 .total phenolic content Folin-Ciocalteu (FCA) reagent method was used to estimate the total phenolic content of the water extracts in the six medicinal plants 26 .The detailed procedure was followed as described 27 .

Total flavonoid content
Aluminum chloride method was used to estimate the total flavonoid content (TFC) of the water extracts of the six medicinal plants 28 .The detailed procedure was followed as described 27 .

Ammonium molybdate dependent antioxidant capacity
Ammonium molybdate dependent total antioxidant capacity of the selected medicinal plant extracts was estimated by 29 .The detailed procedure was followed as described 27 .

DPPH and Hydroxyl radical scavenging activity
DPPH and Hydroxyl radical activity of water extracts were measured as mentioned in detail elsewhere 30,31 .The detailed procedure was followed as described 27 .

Qualitative Phytochemical Screening
The preliminary phytochemical studies on six selected medicinal plants revealed the presence of ten different secondary metabolites.Among the tested components, alkaloids, flavonoids, terpenoids, phenolic, tannins and glycosides showed strong reactions.At the same time, steroids showed very feeble reactions.Anthraquinones showed negative results in the tested plants.

Quantitative estimation of total phenolic/ flavonoid content
The results on total phenolic content in the selected medicinal plants are between 190 to 25 GAE mg/g dwt.The highest TPC is present in TT and the lowest is noticed in CB (Figure 1).The total flavonoid content is between 80 to 12 QE mg/g dwt.The flavonoid content was higher in TT and lowest present in CB (Figure 2).Gallic acid and quercetin were used as standards.

Total antioxidant capacity (TAC)
The results on TAC of the selected medicinal plant extracts indicate that they expressed 240 to 40 ASE mg/g dwt.The highest TAC was observed in TT and the lowest in CB (Figure 3).Ascorbic acid was used as a standard component.

DPPH scavenging activity
The results revealed that all the plants expressed concentration dependent DPPH quenching activity.The plants, AA, CS and MC reduced DPPH purple color strongly by expressing ~80% as maximum inhibition.The extracts exhibited IC 50 values to inhibit 50% of the DPPH radical between 300 to 25 µg/ml (Figure 4).

Hydroxyl radical scavenging activity
All the plants tested exhibited concentration dependent hydroxyl radical scavenging activity (Fig. 5).Among the test plants A. alinifolia showed lowest IC 50 value (36.4 ug/ml) and T. tomentosa showed ~96% as maximum inhibition (Figure 5).

DiSCuSSion
Recently researchers have been interested in exploring natural antioxidant principles that are therapeutically potent and with minimum low or no side effects to treat various human ailments and for the food industry.Plants synthesize a wide variety of chemical components such as alkaloids (nitrogenous compounds), terpenes (lipid derivatives) and phenolics (carbohydrate derivatives) with potential pharmacological properties 32 .Thousands of biologically active phytoconstituents are isolated from higher plants.Of which phenolic compounds are phenolic acids, vitamin E, coumarins, flavonoids, isocoumarins, biflavonols, stilbene, phenols, quinones, betacitie, and chromones etc. reported exhibiting intense antioxidant activity.Scientifically phenolic and flavonoids are reported to have various pharmacological activities such as antioxidant, antiulcer, antispasmodic, cytotoxic antiinflammatory, antitumor, and antidepressant activities 33 to 37 .
The Quantity of total phenolic and flavonoids of the water extracts of the selected medicinal plants is estimated using the colorimetric method.The results indicated that T. tomentosa stem bark has more phenolic and flavonoid contents.The Genus Terminalia belongs to the Combretaceae and comprises about 250 species.Of these, only 39 species were studied for its phytochemical composition and 368 phyto-constituents such as terpenoids, flavonoids, tannins, simple phenolics, phenylpropanoids, etc. Quantitative estimation of phenolic and flavonoid content from T. tomentosa stem bark was reported 38 .They reported higher values of TPC and less TFC than the contents of the present study.Budholiya and Sharma 39 estimated total phenolic and flavonoid contents from T. toemntosa leaf extracts.
Vasthi Kennedy and Devarajan Natarajan 40 studied antioxidant and phytochemical analysis of A. alnifolia leaf, especially methanol and aqueous extracts.In the present study, water extract has higher phenolic and flavonoid content than the previous report (Fig. 2A & B).TPC and TFC of C. bonduc seeds showed a lower range than previously reported results 41 .This may be due to the variation in the climatic condition in both atmospheric and edaphic conditions.
Phytochemical reports of the genus Commiphora, resulted in the identification of more than 300 chemical constituents 42 .Very few and sporadic attempts were made on phenolic and flavonoid contents of C. caudata leaf ethanol extract 43 .M. concanensis a rare wild medicinal plant of tropical deciduous forest, received good attention in phytochemical reports of different parts.The results indicated the presence of volatile oils, flavonoids, alkaloids, tannins, and fatty acids

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. Few reports were noticed on quantitative analysis of phenolics and flavonoids from M. concanensis 45 .A critical review of quantitative estimation of the phytochemical composition of aqueous extracts of C. caudata (stem bark), C. spinarum (fruits), and M. concanensis (leaves) revealed that no previous report was noticed on total phenolic and flavonoid contents, hence, present report on the said species gains importance.The standard and common method used to estimate the antioxidant capacity of medicinal plant extract is the phosphomolybdenum method.Antioxidant components reduce molybdenum and form a green coloured MO V complex.Revathi et al., 8 reported phosphomolybdenum dependent antioxidant capacity of A. alnifolia leaf extracts i.e., Petroleum ether (38.7 ± 2.2), methanol (139.7 ± 2.8) and hot water extract (82.9 ± 6.4 mg AAE/g).In our current study the total antioxidant capacity of water extract of A. alnifolia aerial parts showed similar values (80.13 ± 1.05 mg AAE/g) to the previously reported values.Based on the review of the antioxidant potential of aqueous extracts on ammonium molybdate dependent antioxidant capacity of C. caudata (stem bark), C. spinarum (fruits), M. concanensis (leaves) and T. tomentosa (stem bark) revealed that no previous report was noticed on ammonium molybdate dependent antioxidant capacity.Hence, the present report on the said species gains importance.
DPPH is a synthetic free radical commonly used to evaluate the antioxidant potential of herbal drugs/pure constituents.The results on DPPH reducing activity revealed that, among the test plant extracts, A. alnifolia strongly (88%) reduced DPPH purple color than other plant extracts.T. tomentosa exhibited the lowest IC 50 value (25 µg/ ml), indicating its potential as an antioxidant.The intense DPPH quenching activity of T. tomentosa may be responsible for the presence of ellagic acid and gallic acid, which were reported as potential antioxidants/ therapeutics from different traditional medicinal plant sources 46 .Evangelene and Natarajan 47 observed the antioxidant activity of A. alnifolia leaf methanol and water extracts by the DPPH method.Here, we studied the DPPH scavenging activity of water extract of A. alnifolia aerial parts, the results demonstrate that water exhibited IC 50 values (42.5µg/ml) higher than leaf extracts.DPPH reducing activity of C. caudata stem bark extracts ethyl acetate, methanol, petroleum ether and chloroform was reported 48 .
Here we reported DPPH quenching capacity of water extract.Vijay Kumar et al., 49 reported DPPH scavenging activity of methanol extract of M. concanensis leaf.The results indicated that ethanol and methanol extracts showed maximum inhibition of 69% and 66 % at 250 µg/ml concentration.In the present study, also reported the DPPH quenching capacity of water extract.It required 60 µg/ ml to reduce 50% DPPH purple color.A critical review of the literature indicated that no previous report was noticed on the DPPH scavenging activity of water extract of C. caudata (stem bark), C. spinarum (fruits), P. toemntosa (leaves) and T. toemntosa (stem bark).
Hydroxyl radicals formed through Fenton's reaction are the most reactive oxidative molecules capable of damaging biological molecules/ membranes in living cells.In the present experiment, T. tomentosa exhibited maximum hydroxyl radical inhibition as ~96%.A. alnifolia showed the lowest IC 50 value (36.4 µg/ ml), indicating its potentiality as hydroxyl radical inhibitor.Free radical scavenging property of C. caudata stem bark was reported by DPPH, nitric oxide, SOD methods by in vitro 43,48,50 and in-vivo studies 51 .Antioxidant activity of leaf and fruit oils of C. caudata was reported by Reddy et al., 52 .
No previous report was noticed on the hydroxyl radical scavenging capacity of C. caudata stem bark extracts by in vitro studies.
The Hydroxyl radical quenching activity of M. concanensis leaf ethanol extract was studied by Balakrishnan & Krishnasamy 53 .The results showed that, ethanol extract expressed very feeble activity i.e. it expressed a very high IC 50 value (400 µg/ ml) and 70% as maximum inhibition at 500 µg/ ml concentration.In the present study, water extract exhibited significant hydroxyl radical scavenging activity by expressing a low IC 50 value i.e., 60 µg/ ml.Critical review on hydroxyl radical scavenging activity of A. alnifolia aerial parts 54 , C. spinarum (fruits), T. tomentosa stem bark 55,56 , and P. tomentosa leaves 57 was not reported by earlier workers.Hence, the present work provides additional information on the hydroxyl radical scavenging potential of above mentioned plants.

ConCluSion
The current in vitro studies indicated that water extracts of the selected medicinal plants have a high amount of phenolic and flavonoid components, good antioxidant capacity and strongly inhibited Hydroxyl radicals and reduced DPPH, The findings of the study suggest that the selected medicinal plants could be used as a potential source of natural antioxidants.

Conflict of interest
Authors declared as no conflict of interest.

Funding Sources
Nil.

table 1 .
Qualitative phytochemical analysis of water extracts of selected medicinal plants Fig. 1.Photographs of the selected medicinal plants